Plants Having Enhanced Yield-Related Traits and a Method for Making the Same

ABSTRACT

The present invention relates generally to the field of molecular biology and concerns a method for enhancing various economically important yield-related traits in plants. More specifically, the present invention concerns a method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding an ASPAT (Asparatate AminoTransferase) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding an ASPAT polypeptide, which plants have enhanced yield-related traits relative to control plants. The invention also provides hitherto unknown ASPAT-encoding nucleic acids and constructs comprising the same, useful in performing the methods of the invention. Furthermore, the present invention relates generally to the field of molecular biology and concerns a method for increasing various plant yield-related traits by increasing expression in a plant of a nucleic acid sequence encoding a MYB91 like transcription factor (MYB91) polypeptide. The present invention also concerns plants having increased expression of a nucleic acid sequence encoding an MYB91 polypeptide, which plants have increased yield-related traits relative to control plants. The invention additionally relates to nucleic acid sequences, nucleic acid constructs, vectors and plants containing said nucleic acid sequences. Even furthermore, the present invention relates generally to the field of molecular biology and concerns a method for improving various plant growth characteristics by modulating expression in a plant of a nucleic acid encoding a GASA (Gibberellic Acid-Stimulated  Arabidopsis ). The present invention also concerns plants having modulated expression of a nucleic acid encoding a GASA, which plants have improved growth characteristics relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention. Yet furthermore, the present invention relates generally to the field of molecular biology and concerns a method for enhancing various economically important yield-related traits in plants. More specifically, the present invention concerns a method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding an AUX/IAA (auxin/indoleacetic acid) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding IAA polypeptide, which plants have enhanced yield-related traits relative to control plants. The invention also provides constructs comprising AUX/IAA-encoding nucleic acids, useful in performing the methods of the invention.

The present invention relates generally to the field of molecularbiology and concerns a method for enhancing various economicallyimportant yield-related traits in plants. More specifically, the presentinvention concerns a method for enhancing yield-related traits in plantsby modulating expression in a plant of a nucleic acid encoding an ASPAT(Asparatate AminoTransferase) polypeptide. The present invention alsoconcerns plants having modulated expression of a nucleic acid encodingan ASPAT polypeptide, which plants have enhanced yield-related traitsrelative to control plants. The invention also provides hitherto unknownASPAT-encoding nucleic acids and constructs comprising the same, usefulin performing the methods of the invention.

Furthermore, the present invention relates generally to the field ofmolecular biology and concerns a method for increasing various plantyield-related traits by increasing expression in a plant of a nucleicacid sequence encoding a MYB91 like transcription factor (MYB91)polypeptide. The present invention also concerns plants having increasedexpression of a nucleic acid sequence encoding an MYB91 polypeptide,which plants have increased yield-related traits relative to controlplants. The invention additionally relates to nucleic acid sequences,nucleic acid constructs, vectors and plants containing said nucleic acidsequences.

Even furthermore, the present invention relates generally to the fieldof molecular biology and concerns a method for improving various plantgrowth characteristics by modulating expression in a plant of a nucleicacid encoding a GASA (Gibberellic Acid-Stimulated Arabidopsis). Thepresent invention also concerns plants having modulated expression of anucleic acid encoding a GASA, which plants have improved growthcharacteristics relative to corresponding wild type plants or othercontrol plants. The invention also provides constructs useful in themethods of the invention.

Yet furthermore, the present invention relates generally to the field ofmolecular biology and concerns a method for enhancing variouseconomically important yield-related traits in plants. Morespecifically, the present invention concerns a method for enhancingyield-related traits in plants by modulating expression in a plant of anucleic acid encoding an AUX/IAA (auxin/indoleacetic acid) polypeptide.The present invention also concerns plants having modulated expressionof a nucleic acid encoding IAA polypeptide, which plants have enhancedyield-related traits relative to control plants. The invention alsoprovides constructs comprising AUX/IAA-encoding nucleic acids, useful inperforming the methods of the invention.

The ever-increasing world population and the dwindling supply of arableland available for agriculture fuels research towards increasing theefficiency of agriculture. Conventional means for crop and horticulturalimprovements utilise selective breeding techniques to identify plantshaving desirable characteristics. However, such selective breedingtechniques have several drawbacks, namely that these techniques aretypically labour intensive and result in plants that often containheterogeneous genetic components that may not always result in thedesirable trait being passed on from parent plants. Advances inmolecular biology have allowed mankind to modify the germplasm ofanimals and plants. Genetic engineering of plants entails the isolationand manipulation of genetic material (typically in the form of DNA orRNA) and the subsequent introduction of that genetic material into aplant. Such technology has the capacity to deliver crops or plantshaving various improved economic, agronomic or horticultural traits.

A trait of particular economic interest is increased yield. Yield isnormally defined as the measurable produce of economic value from acrop. This may be defined in terms of quantity and/or quality. Yield isdirectly dependent on several factors, for example, the number and sizeof the organs, plant architecture (for example, the number of branches),seed production, leaf senescence and more. Root development, nutrientuptake, stress tolerance and early vigour may also be important factorsin determining yield. Optimizing the abovementioned factors maytherefore contribute to increasing crop yield.

Seed yield is a particularly important trait, since the seeds of manyplants are important for human and animal nutrition. Crops such as corn,rice, wheat, canola and soybean account for over half the total humancaloric intake, whether through direct consumption of the seedsthemselves or through consumption of meat products raised on processedseeds. They are also a source of sugars, oils and many kinds ofmetabolites used in industrial processes. Seeds contain an embryo (thesource of new shoots and roots) and an endosperm (the source ofnutrients for embryo growth during germination and during early growthof seedlings). The development of a seed involves many genes, andrequires the transfer of metabolites from the roots, leaves and stemsinto the growing seed. The endosperm, in particular, assimilates themetabolic precursors of carbohydrates, oils and proteins and synthesizesthem into storage macromolecules to fill out the grain.

Plant biomass is yield for forage crops like alfalfa, silage corn andhay. Many proxies for yield have been used in grain crops. Chief amongstthese are estimates of plant size. Plant size can be measured in manyways depending on species and developmental stage, but include totalplant dry weight, above-ground dry weight, above-ground fresh weight,leaf area, stem volume, plant height, rosette diameter, leaf length,root length, root mass, tiller number and leaf number. Many speciesmaintain a conservative ratio between the size of different parts of theplant at a given developmental stage. These allometric relationships areused to extrapolate from one of these measures of size to another (e.g.Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant size at anearly developmental stage will typically correlate with plant size laterin development. A larger plant with a greater leaf area can typicallyabsorb more light and carbon dioxide than a smaller plant and thereforewill likely gain a greater weight during the same period (Fasoula &Tollenaar 2005 Maydica 50:39). This is in addition to the potentialcontinuation of the micro-environmental or genetic advantage that theplant had to achieve the larger size initially. There is a stronggenetic component to plant size and growth rate (e.g. ter Steege et al2005 Plant Physiology 139:1078), and so for a range of diverse genotypesplant size under one environmental condition is likely to correlate withsize under another (Hittalmani et al 2003 Theoretical Applied Genetics107:679). In this way a standard environment is used as a proxy for thediverse and dynamic environments encountered at different locations andtimes by crops in the field.

Another important trait for many crops is early vigour. Improving earlyvigour is an important objective of modern rice breeding programs inboth temperate and tropical rice cultivars. Long roots are important forproper soil anchorage in water-seeded rice. Where rice is sown directlyinto flooded fields, and where plants must emerge rapidly through water,longer shoots are associated with vigour. Where drill-seeding ispracticed, longer mesocotyls and coleoptiles are important for goodseedling emergence. The ability to engineer early vigour into plantswould be of great importance in agriculture. For example, poor earlyvigour has been a limitation to the introduction of maize (Zea mays L.)hybrids based on Corn Belt germplasm in the European Atlantic.

Harvest index, the ratio of seed yield to aboveground dry weight, isrelatively stable under many environmental conditions and so a robustcorrelation between plant size and grain yield can often be obtained(e.g. Rebetzke et al 2002 Crop Science 42:739). These processes areintrinsically linked because the majority of grain biomass is dependenton current or stored photosynthetic productivity by the leaves and stemof the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa StateUniversity Press, pp 68-73). Therefore, selecting for plant size, evenat early stages of development, has been used as an indicator for futurepotential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105:213). When testing for the impact of genetic differences on stresstolerance, the ability to standardize soil properties, temperature,water and nutrient availability and light intensity is an intrinsicadvantage of greenhouse or plant growth chamber environments compared tothe field. However, artificial limitations on yield due to poorpollination due to the absence of wind or insects, or insufficient spacefor mature root or canopy growth, can restrict the use of thesecontrolled environments for testing yield differences. Therefore,measurements of plant size in early development, under standardizedconditions in a growth chamber or greenhouse, are standard practices toprovide indication of potential genetic yield advantages.

A further important trait is that of improved abiotic stress tolerance.Abiotic stress is a primary cause of crop loss worldwide, reducingaverage yields for most major crop plants by more than 50% (Wang et al.,Planta (2003) 218: 1-14). Abiotic stresses may be caused by drought,salinity, extremes of temperature, chemical toxicity and oxidativestress. The ability to improve plant tolerance to abiotic stress wouldbe of great economic advantage to farmers worldwide and would allow forthe cultivation of crops during adverse conditions and in territorieswhere cultivation of crops may not otherwise be possible.

Crop yield may therefore be increased by optimising one of theabove-mentioned factors.

Depending on the end use, the modification of certain yield traits maybe favoured over others. For example for applications such as forage orwood production, or bio-fuel resource, an increase in the vegetativeparts of a plant may be desirable, and for applications such as flour,starch or oil production, an increase in seed parameters may beparticularly desirable. Even amongst the seed parameters, some may befavoured over others, depending on the application. Various mechanismsmay contribute to increasing seed yield, whether that is in the form ofincreased seed size or increased seed number.

One approach to increasing yield (seed yield and/or biomass) in plantsmay be through modification of the inherent growth mechanisms of aplant, such as the cell cycle or various signalling pathways involved inplant growth or in defense mechanisms.

Concerning ASPAT polypeptides, it has now been found that variousyield-related traits may be improved in plants by modulating expressionin a plant of a nucleic acid encoding an ASPAT (AspartateAminoTransferase) in a plant.

Concerning MYB91 polypeptides, it has now been found that variousyield-related traits may be increased in plants relative to controlplants, by increasing expression in a plant of a nucleic acid sequenceencoding a MYB91 like transcription factor (MYB91) polypeptide. Theincreased yield-related traits comprise one or more of: increased plantheight, increased harvest index (HI), and increased Thousand KernelWeight (TKW).

Concerning GASA polypeptides, it has now been found that various growthcharacteristics may be improved in plants by modulating expression in aplant of a nucleic acid encoding a GASA (Gibberellic Acid-StimulatedArabidopsis) in a plant.

Concerning AUX/IAA polypeptides it has now been found that variousgrowth characteristics may be improved in plants by modulatingexpression in a plant of a nucleic acid encoding an AUX/IAA polypeptidein a plant.

BACKGROUND

1. Aspartate AminoTransferase (ASPAT)

The capacity for growth, development and yield production of a plant isinfluenced by the regulation of carbon and nitrogen metabolisms and theN/C ratio in a the plant Lawlor 2002 Journal of Experimental Botany,Vol. 53, No. 370, pp. 773-787.

The enzyme Aspartate aminotransferase (ASPAT enzyme) catalyzes catalysesthe reversible reaction of transamination between aspartate and2-oxoglutarate to generate glutamate and oxaloacetate using pyridoxal5¢-phosphate (PLP) as essential cofactor in a reaction that can beexpress as: L-aspartate+2-oxoglutarate=oxaloacetate+L-glutamate.

The enzyme plays a key role in the metabolic regulation of carbon andnitrogen metabolism in all organisms. Structurally and functionally theASPAT enzyme is conserved in all organisms. In eukaryots the enzymeplays a critical role in the interchanges of carbon and nitrogen poolsbetween subcellular compartments.

Aspartate aminotransferases are classified into the group I of theaminotransferase superfamily (Jensen and Gu, 1996). Further, AspartateAminotransferases have been classified in four subgroups. Subgroup Iaincludes the ASPATs from eubacteria and eukaryotes, whereas subgroup Ibcomprises the enzymes from some eubacteria including cyanobacteria andarchaebacteria. A new group of ASPAT enzymes was described by De LaTorre et al. 2006 Plant J. 2006, 46(3):414-25.

In plants, genes have been identified encoding ASPAT polypeptides thatare targeted to different subcellular compartments and assembled intofunctional ASPAT Isoenzymes in the mitochondria, the cytosol, theperoxisome and the chloroplast.

2. MYB91 Like Transcription Factor (MYB91)

DNA-binding proteins are proteins that comprise any of many DNA-bindingdomains and thus have a specific or general affinity to DNA. DNA-bindingproteins include for example transcription factors that modulate theprocess of transcription, nucleases that cleave DNA molecules, andhistones that are involved in DNA packaging in the cell nucleus.

Transcription factors are usually defined as proteins that showsequence-specific DNA binding affinity and that are capable ofactivating and/or repressing transcription. The Arabidopsis thalianagenome codes for at least 1533 transcriptional regulators, accountingfor ˜5.9% of its estimated total number of genes (Riechmann et al.(2000) Science 290: 2105-2109). The Database of Rice TranscriptionFactors (DRTF) is a collection of known and predicted transcriptionfactors of Oryza sativa L. ssp. indica and Oryza sativa L. ssp.japonica, and currently contains 2,025 putative transcription factors(TF) gene models in indica and 2,384 in japonica, distributed in 63families (Gao et al. (2006) Bioinformatics 2006, 22(10):1286-7).

One of these families is the MYB domain family of transcription factors,characterized by a highly conserved DNA-binding domain, the MYB domain.The MYB domain was originally described in the oncogene (v-myb) of avianmyeloblastosis virus (Klempnauer et al. (1982) Cell 33, 453-63). Manyvertebrates contain three genes related to v-Myb c-Myb, A-Myb and B-Myband other similar genes have been identified in insects, plants, fungiand slime molds. The encoded proteins are crucial to the control ofproliferation and differentiation in a number of cell types. MYBproteins contain one to four imperfect direct repeats of a conservedsequence of 50-53 amino acids which encodes a helix-turn-helix structureinvolved in DNA binding (Rosinski and Atchley (1998) J Mol Evol 46,74-83). Three regularly spaced tryptophan residues, which form atryptophan cluster in the three-dimensional helix-turn-helix structure,are characteristic of a MYB repeat. The three repeats in c-Myb arereferred to as R1, R2 and R3; and repeats from other MYB proteins arecategorised according to their similarity to R1, R2 or R3. Since thereis limited sequence conservation outside of the MYB domain, MYB proteinshave been clustered into subgroups based on conserved motifs identifiedoutside of the MYB coding region (Jiang et al. (2004) Genome Biology 5,R46).

AtMYB91 belongs to the R2R3-MYB gene family (Li and Parish, Plant J. 8,963-972, 1995), which is a large gene family (with reportedly 126 genesin Arabidopsis thaliana (Zimmerman et al., Plant J. 40, 22-34, 2004)).Members of this group are involved in various processes, includingsecondary metabolism, cell morphogenesis, regulation of meristemformation, flower and seed development, cell cycle, defense and stressresponses, light and hormone signalling (Chen et al., Cell Res. 16,797-798, 2006). AtMYB91 is also named AS1 asymmetric leaves 1, and isclosely related to Antirrhinum PHAN phantastica and to maize ROUGHSHEATH2 (RS2) polypeptides (Sun et al. (2002) Planta 214(5):694-702),all having an evolutionarily conserved role in specification of leafcell identity, in particular in dorsal-ventral identity. In Arabidopsis,AS1 is expressed in leaf founder cells, where it functions as aheterodimer with the structurally unrelated AS2 proteins to repressactivity of KNOTTED 1-like homeobox (KNOX) genes.

3. Gibberellic Acid-Stimulated Arabidopsis (GASA)

GASA (Gibberellic Acid-Stimulated Arabidopsis) proteins areplant-specific and are expressed during a variety of physiologicalprocesses. Several GASA-like genes are hormone responsive, expression oftomato gene GAST1, the first member of the family to be characterized,was induced upon application of exogenous gibberellin in agibberellin-deficient background (Shi et al. Plant J. 2, 153-159, 1992).A related tomato gene, RSI-1, shares high sequence identity with GAST1and is activated during lateral root formation (Taylor and Scheuring,Mol. Gen. Genet. 243, 148-157, 1994). GASA1 to GASA4 from Arabidopsiswere first identified based on their similarity to tomato GAST1 (Herzoget al. Plant Mol. Biol. 27, 743-752, 1995). Expression data indicatedthat GASA1 accumulates in flower buds and immature siliques, GASA2 andGASA3 in siliques and dry seeds, and GASA4 in growing roots and flowerbuds. GASA4 is reported to be expressed in all meristematic regions(Aubert et al., Plant Mol. Biol. 36, 871-883, 1998).

Functionally, the GASA proteins are not well characterised. GASAproteins are reportedly involved in pathogen responses and in plantdevelopment. Plants ectopically expressing GEG, a GASA homologue fromGerbera hybrida, showed shorter corollas with decreased cell lengthcompared with the wild type, indicating a role for GEG as an inhibitorof cell elongation. Overexpression of Arabidopsis GASA4 resulted inplants having increased seed weight (Roxrud et al, Plant Cell Physiol.48, 471-483, 2007). However, these plants in addition had occasionalmeristem identity changes with reconversion from floral meristemsdevelopment to normal indeterminate inflorescence development.Furthermore, modulated GASA4 expression caused a significant increase ofbranching. Overexpression of Arabidopsis GASA4 also increased toleranceto heat stress (Ko et al., Plant Physiol. Biochem. 45, 722-728, 2007).

4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

The AUX/IAA (auxin/indoleacetic acid) genes encode a family of proteinswhose expression is tightly regulated by auxin. The plant hormone auxinis involved in various processes like cell division, cell expansion anddifferentiation, patterning of embryos, vasculature or other tissues,regulation of growth of primary and lateral root or shoot meristems.AUX/IAA proteins furthermore are usually expressed in a tissue-specificmanner.

AUX/IAA proteins typically have four conserved amino acid sequencemotifs (domains I, II, III and IV) and have nuclear localisation signalsequences. Domains I and II are postulated to destabilize the proteinand may be involved in protein turnover. Domains III and IV arepostulated to be involved in protein-protein interactions: AUX/IAAproteins can form homodimers and are known to associate with ARFproteins. The AUX/IAA-ARF complexes are likely to be involved in auxinmediated gene expression. The Aux/IAA proteins are negative regulatorsof the auxin response factors (ARFs) that regulate expression ofauxin-responsive genes. Aux/IAA proteins bind to the DNA-bound ARFpartner proteins and repress ARF activity. In the auxin activatedstatus, Aux/IAA proteins are ubiquitinated via interactions with theauxin-modified SCFTIR1complex and subsequently degraded by 26Sproteasome action. An overview of roles and activities of AUX/IAAproteins is given by Reed (Trends in Plant Science 6, 420-425, 2001).The structure and expression analysis of early auxin-responsive Aux/IAAgene family in rice (Oryza sativa) has recently been reported by Jain etal. 2006 Funct Integr Genomics. 2006 January; 6(1):47-59.

IAA14 is a AUX/IAA protein that acts as a transcriptional repressor inlateral root formation. A gain of function mutation in IAA14 blocksearly pericycle divisions that initiate lateral root development (Fukakiet al., Plant J. 29, 153-168, 2002).

SUMMARY 1. Aspartate AminoTransferase (ASPAT)

Surprisingly, it has now been found that modulating expression of anucleic acid encoding an ASPAT polypeptide gives plants having enhancedyield-related traits relative to control plants.

According one embodiment, there is provided a method for enhancingyield-related traits relative to control plants, comprising modulatingexpression of a nucleic acid encoding an ASPAT polypeptide in a plant.

2. MYB91 Like Transcription Factor (MYB91)

Surprisingly, it has now been found that increasing expression in aplant of a nucleic acid sequence encoding a MYB91 like transcriptionfactor (MYB91) polypeptide as defined herein, gives plants havingincreased yield-related traits relative to control plants.

According to one embodiment, there is provided a method for increasingyield-related traits in plants relative to control plants, comprisingincreasing expression in a plant of a nucleic acid sequence encoding aMYB91 like transcription factor (MYB91) as defined herein. The increasedyield-related traits comprise one or more of: increased plant height,increased harvest index (HI), and increased Thousand Kernel Weight(TKW).

3. Gibberellic Acid-Stimulated Arabidopsis (GASA)

Surprisingly, it has now been found that modulating expression of anucleic acid encoding a GASA polypeptide gives plants having enhancedyield-related traits, in particular increased yield relative to controlplants.

According one embodiment, there is provided a method for improving yieldrelated traits of a plant, relative to control plants, comprisingmodulating expression of a nucleic acid encoding a GASA polypeptide in aplant.

4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

Surprisingly, it has now been found that modulating expression of anucleic acid encoding an AUX/IAA polypeptide gives plants havingenhanced yield-related traits, in particular increased yield relative tocontrol plants.

According one embodiment, there is provided a method for improving yieldrelated traits of a plant relative to control plants, comprisingmodulating expression of a nucleic acid encoding an AUX/IAA polypeptidein a plant, wherein the yield related traits do not encompass increasedroot growth.

DEFINITIONS Polypeptide(s)/Protein(s)

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to amino acids in a polymeric form of any length, linkedtogether by peptide bonds.

Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/NucleotideSequence(s)

The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotidesequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are usedinterchangeably herein and refer to nucleotides, either ribonucleotidesor deoxyribonucleotides or a combination of both, in a polymericunbranched form of any length.

Control Plant(s)

The choice of suitable control plants is a routine part of anexperimental setup and may include corresponding wild type plants orcorresponding plants without the gene of interest. The control plant istypically of the same plant species or even of the same variety as theplant to be assessed. The control plant may also be a nullizygote of theplant to be assessed. Nullizygotes are individuals missing the transgeneby segregation. A “control plant” as used herein refers not only towhole plants, but also to plant parts, including seeds and seed parts.

Homologue(s)

“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar biological and functional activity as theunmodified protein from which they are derived.

A deletion refers to removal of one or more amino acids from a protein.

An insertion refers to one or more amino acid residues being introducedinto a predetermined site in a protein. Insertions may compriseN-terminal and/or C-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Generally, insertionswithin the amino acid sequence will be smaller than N- or C-terminalfusions, of the order of about 1 to 10 residues. Examples of N- orC-terminal fusion proteins or peptides include the binding domain oractivation domain of a transcriptional activator as used in the yeasttwo-hybrid system, phage coat proteins, (histidine)-6-tag, glutathioneS-transferase-tag, protein A, maltose-binding protein, dihydrofolatereductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP(calmodulin-binding peptide), HA epitope, protein C epitope and VSVepitope.

A substitution refers to replacement of amino acids of the protein withother amino acids having similar properties (such as similarhydrophobicity, hydrophilicity, antigenicity, propensity to form orbreak α-helical structures or β-sheet structures). Amino acidsubstitutions are typically of single residues, but may be clustereddepending upon functional constraints placed upon the polypeptide;insertions will usually be of the order of about 1 to 10 amino acidresidues. The amino acid substitutions are preferably conservative aminoacid substitutions. Conservative substitution tables are well known inthe art (see for example Creighton (1984) Proteins. W.H. Freeman andCompany (Eds) and Table 1 below).

TABLE 1 Examples of conserved amino acid substitutions ResidueConservative Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln AsnCys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg;Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr TyrTrp; Phe Val Ile; Leu

Amino acid substitutions, deletions and/or insertions may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulation. Methods for the manipulation of DNA sequences to producesubstitution, insertion or deletion variants of a protein are well knownin the art. For example, techniques for making substitution mutations atpredetermined sites in DNA are well known to those skilled in the artand include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, SanDiego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols.

Derivatives

“Derivatives” include peptides, oligopeptides, polypeptides which may,compared to the amino acid sequence of the naturally-occurring form ofthe protein, such as the protein of interest, comprise substitutions ofamino acids with non-naturally occurring amino acid residues, oradditions of non-naturally occurring amino acid residues. “Derivatives”of a protein also encompass peptides, oligopeptides, polypeptides whichcomprise naturally occurring altered (glycosylated, acylated,prenylated, phosphorylated, myristoylated, sulphated etc.) ornon-naturally altered amino acid residues compared to the amino acidsequence of a naturally-occurring form of the polypeptide. A derivativemay also comprise one or more non-amino acid substituents or additionscompared to the amino acid sequence from which it is derived, forexample a reporter molecule or other ligand, covalently ornon-covalently bound to the amino acid sequence, such as a reportermolecule which is bound to facilitate its detection, and non-naturallyoccurring amino acid residues relative to the amino acid sequence of anaturally-occurring protein. Furthermore, “derivatives” also includefusions of the naturally-occurring form of the protein with taggingpeptides such as FLAG, HIS6 or thioredoxin (for a review of taggingpeptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).

Orthologue(s)/Paralogue(s)

Orthologues and paralogues encompass evolutionary concepts used todescribe the ancestral relationships of genes. Paralogues are geneswithin the same species that have originated through duplication of anancestral gene; orthologues are genes from different organisms that haveoriginated through speciation, and are also derived from a commonancestral gene.

Domain

The term “domain” refers to a set of amino acids conserved at specificpositions along an alignment of sequences of evolutionarily relatedproteins. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely essential in the structure,stability or function of a protein. Identified by their high degree ofconservation in aligned sequences of a family of protein homologues,they can be used as identifiers to determine if any polypeptide inquestion belongs to a previously identified polypeptide family.

Motif/Consensus sequence/Signature

The term “motif” or “consensus sequence” or “signature” refers to ashort conserved region in the sequence of evolutionarily relatedproteins. Motifs are frequently highly conserved parts of domains, butmay also include only part of the domain, or be located outside ofconserved domain (if all of the amino acids of the motif fall outside ofa defined domain).

Hybridisation

The term “hybridisation” as defined herein is a process whereinsubstantially homologous complementary nucleotide sequences anneal toeach other. The hybridisation process can occur entirely in solution,i.e. both complementary nucleic acids are in solution. The hybridisationprocess can also occur with one of the complementary nucleic acidsimmobilised to a matrix such as magnetic beads, Sepharose beads or anyother resin.

The hybridisation process can furthermore occur with one of thecomplementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to, for example, a siliceous glass support (the latterknown as nucleic acid arrays or microarrays or as nucleic acid chips).In order to allow hybridisation to occur, the nucleic acid molecules aregenerally thermally or chemically denatured to melt a double strand intotwo single strands and/or to remove hairpins or other secondarystructures from single stranded nucleic acids.

The term “stringency” refers to the conditions under which ahybridisation takes place. The stringency of hybridisation is influencedby conditions such as temperature, salt concentration, ionic strengthand hybridisation buffer composition. Generally, low stringencyconditions are selected to be about 30° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. Medium stringency conditions are when the temperatureis 20° C. below T_(m), and high stringency conditions are when thetemperature is 10° C. below T_(m). High stringency hybridisationconditions are typically used for isolating hybridising sequences thathave high sequence similarity to the target nucleic acid sequence.However, nucleic acids may deviate in sequence and still encode asubstantially identical polypeptide, due to the degeneracy of thegenetic code. Therefore medium stringency hybridisation conditions maysometimes be needed to identify such nucleic acid molecules.

The Tm is the temperature under defined ionic strength and pH, at which50% of the target sequence hybridises to a perfectly matched probe. TheT_(m) is dependent upon the solution conditions and the base compositionand length of the probe. For example, longer sequences hybridisespecifically at higher temperatures. The maximum rate of hybridisationis obtained from about 16° C. up to 32° C. below T_(m). The presence ofmonovalent cations in the hybridisation solution reduce theelectrostatic repulsion between the two nucleic acid strands therebypromoting hybrid formation; this effect is visible for sodiumconcentrations of up to 0.4M (for higher concentrations, this effect maybe ignored). Formamide reduces the melting temperature of DNA-DNA andDNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, andaddition of 50% formamide allows hybridisation to be performed at 30 to45° C., though the rate of hybridisation will be lowered. Base pairmismatches reduce the hybridisation rate and the thermal stability ofthe duplexes. On average and for large probes, the Tm decreases about 1°C. per % base mismatch. The Tm may be calculated using the followingequations, depending on the types of hybrids:

-   1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284,    1984):    -   T_(m)=81.5°        C.+16.6×log₁₀[Na⁺]^(a)+0.41×%[G/C^(b)]−500×[L^(c)]⁻¹−0.61×%        formamide-   2) DNA-RNA or RNA-RNA hybrids:    -   Tm=79.8+18.5 (log₁₀[Na⁺]^(a))+0.58 (% G/C^(b))+11.8(%        G/C^(b))²−820/L^(c)-   3) oligo-DNA or oligo-RNAs hybrids:    -   For <20 nucleotides: T_(m)=2 (I_(n))    -   For 20-35 nucleotides: T_(m)=22+1.46 (I_(n))-   ^(a) or for other monovalent cation, but only accurate in the    0.01-0.4 M range.-   ^(b) only accurate for % GC in the 30% to 75% range.-   ^(c)L=length of duplex in base pairs.-   ^(d) oligo, oligonucleotide; I_(n), =effective length of    primer=2×(no. of G/C)+(no. of NT).

Non-specific binding may be controlled using any one of a number ofknown techniques such as, for example, blocking the membrane withprotein containing solutions, additions of heterologous RNA, DNA, andSDS to the hybridisation buffer, and treatment with Rnase. Fornon-homologous probes, a series of hybridizations may be performed byvarying one of (i) progressively lowering the annealing temperature (forexample from 68° C. to 42° C.) or (ii) progressively lowering theformamide concentration (for example from 50% to 0%). The skilledartisan is aware of various parameters which may be altered duringhybridisation and which will either maintain or change the stringencyconditions.

Besides the hybridisation conditions, specificity of hybridisationtypically also depends on the function of post-hybridisation washes. Toremove background resulting from non-specific hybridisation, samples arewashed with dilute salt solutions. Critical factors of such washesinclude the ionic strength and temperature of the final wash solution:the lower the salt concentration and the higher the wash temperature,the higher the stringency of the wash. Wash conditions are typicallyperformed at or below hybridisation stringency. A positive hybridisationgives a signal that is at least twice of that of the background.Generally, suitable stringent conditions for nucleic acid hybridisationassays or gene amplification detection procedures are as set forthabove. More or less stringent conditions may also be selected. Theskilled artisan is aware of various parameters which may be alteredduring washing and which will either maintain or change the stringencyconditions.

For example, typical high stringency hybridisation conditions for DNAhybrids longer than 50 nucleotides encompass hybridisation at 65° C. in1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at65° C. in 0.3×SSC. Examples of medium stringency hybridisationconditions for DNA hybrids longer than 50 nucleotides encompasshybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50%formamide, followed by washing at 50° C. in 2×SSC. The length of thehybrid is the anticipated length for the hybridising nucleic acid. Whennucleic acids of known sequence are hybridised, the hybrid length may bedetermined by aligning the sequences and identifying the conservedregions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate;the hybridisation solution and wash solutions may additionally include5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmentedsalmon sperm DNA, 0.5% sodium pyrophosphate.

For the purposes of defining the level of stringency, reference can bemade to Sambrook et al. (2001) Molecular Cloning: a laboratory manual,3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or toCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989and yearly updates).

Splice Variant

The term “splice variant” as used herein encompasses variants of anucleic acid sequence in which selected introns and/or exons have beenexcised, replaced, displaced or added, or in which introns have beenshortened or lengthened. Such variants will be ones in which thebiological activity of the protein is substantially retained; this maybe achieved by selectively retaining functional segments of the protein.Such splice variants may be found in nature or may be manmade. Methodsfor predicting and isolating such splice variants are well known in theart (see for example Foissac and Schiex (2005) BMC Bioinformatics 6:25).

Allelic Variant

Alleles or allelic variants are alternative forms of a given gene,located at the same chromosomal position. Allelic variants encompassSingle Nucleotide Polymorphisms (SNPs), as well as SmallInsertion/Deletion Polymorphisms (INDELs). The size of INDELs is usuallyless than 100 bp. SNPs and INDELs form the largest set of sequencevariants in naturally occurring polymorphic strains of most organisms.

Gene Shuffling/Directed Evolution

Gene shuffling or directed evolution consists of iterations of DNAshuffling followed by appropriate screening and/or selection to generatevariants of nucleic acids or portions thereof encoding proteins having amodified biological activity (Castle et al., (2004) Science 304(5674):1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).

Regulatory Element/Control Sequence/Promoter

The terms “regulatory element”, “control sequence” and “promoter” areall used interchangeably herein and are to be taken in a broad contextto refer to regulatory nucleic acid sequences capable of effectingexpression of the sequences to which they are ligated. The term“promoter” typically refers to a nucleic acid control sequence locatedupstream from the transcriptional start of a gene and which is involvedin recognising and binding of RNA polymerase and other proteins, therebydirecting transcription of an operably linked nucleic acid. Encompassedby the aforementioned terms are transcriptional regulatory sequencesderived from a classical eukaryotic genomic gene (including the TATA boxwhich is required for accurate transcription initiation, with or withouta CCAAT box sequence) and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner. Also included within the term is atranscriptional regulatory sequence of a classical prokaryotic gene, inwhich case it may include a −35 box sequence and/or −10 boxtranscriptional regulatory sequences. The term “regulatory element” alsoencompasses a synthetic fusion molecule or derivative that confers,activates or enhances expression of a nucleic acid molecule in a cell,tissue or organ.

A “plant promoter” comprises regulatory elements, which mediate theexpression of a coding sequence segment in plant cells. Accordingly, aplant promoter need not be of plant origin, but may originate fromviruses or micro-organisms, for example from viruses which attack plantcells. The “plant promoter” can also originate from a plant cell, e.g.from the plant which is transformed with the nucleic acid sequence to beexpressed in the inventive process and described herein. This alsoapplies to other “plant” regulatory signals, such as “plant”terminators. The promoters upstream of the nucleotide sequences usefulin the methods of the present invention can be modified by one or morenucleotide substitution(s), insertion(s) and/or deletion(s) withoutinterfering with the functionality or activity of either the promoters,the open reading frame (ORF) or the 3′-regulatory region such asterminators or other 3′ regulatory regions which are located away fromthe ORF. It is furthermore possible that the activity of the promotersis increased by modification of their sequence, or that they arereplaced completely by more active promoters, even promoters fromheterologous organisms. For expression in plants, the nucleic acidmolecule must, as described above, be linked operably to or comprise asuitable promoter which expresses the gene at the right point in timeand with the required spatial expression pattern.

For the identification of functionally equivalent promoters, thepromoter strength and/or expression pattern of a candidate promoter maybe analysed for example by operably linking the promoter to a reportergene and assaying the expression level and pattern of the reporter genein various tissues of the plant. Suitable well-known reporter genesinclude for example beta-glucuronidase or beta-galactosidase. Thepromoter activity is assayed by measuring the enzymatic activity of thebeta-glucuronidase or beta-galactosidase. The promoter strength and/orexpression pattern may then be compared to that of a reference promoter(such as the one used in the methods of the present invention).Alternatively, promoter strength may be assayed by quantifying mRNAlevels or by comparing mRNA levels of the nucleic acid used in themethods of the present invention, with mRNA levels of housekeeping genessuch as 18S rRNA, using methods known in the art, such as Northernblotting with densitometric analysis of autoradiograms, quantitativereal-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).Generally by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By “low level” isintended at levels of about 1/10,000 transcripts to about 1/100,000transcripts, to about 1/500,0000 transcripts per cell. Conversely, a“strong promoter” drives expression of a coding sequence at high level,or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000transcripts per cell. Generally, by “medium strength promoter” isintended a promoter that drives expression of a coding sequence at alower level than a strong promoter, in particular at a level that is inall instances below that obtained when under the control of a 35S CaMVpromoter.

Operably Linked

The term “operably linked” as used herein refers to a functional linkagebetween the promoter sequence and the gene of interest, such that thepromoter sequence is able to initiate transcription of the gene ofinterest.

Constitutive Promoter

A “constitutive promoter” refers to a promoter that is transcriptionallyactive during most, but not necessarily all, phases of growth anddevelopment and under most environmental conditions, in at least onecell, tissue or organ. Table 2a below gives examples of constitutivepromoters.

TABLE 2a Examples of constitutive promoters Gene Source Reference ActinMcElroy et al, Plant Cell, 2: 163-171, 1990 HMGP WO 2004/070039 CAMV 35SOdell et al, Nature, 313: 810-812, 1985 CaMV 19S Nilsson et al.,Physiol. Plant. 100: 456-462, 1997 GOS2 de Pater et al, Plant J Nov;2(6): 837-44, 1992, WO 2004/065596 Ubiquitin Christensen et al, PlantMol. Biol. 18: 675-689, 1992 Rice cyclophilin Buchholz et al, Plant MolBiol. 25(5): 837-43, 1994 Maize H3 histone Lepetit et al, Mol. Gen.Genet. 231: 276-285, 1992 Alfalfa H3 Wu et al. Plant Mol. Biol. 11:641-649, 1988 histone Actin 2 An et al, Plant J. 10(1); 107-121, 199634S FMV Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443 Rubiscosmall U.S. Pat. No. 4,962,028 subunit OCS Leisner (1988) Proc Natl AcadSci USA 85(5): 2553 SAD1 Jain et al., Crop Science, 39 (6), 1999: 1696SAD2 Jain et al., Crop Science, 39 (6), 1999: 1696 nos Shaw et al.(1984) Nucleic Acids Res. 12(20): 7831-7846 V-ATPase WO 01/14572 Superpromoter WO 95/14098 G-box proteins WO 94/12015

Ubiquitous Promoter

A ubiquitous promoter is active in substantially all tissues or cells ofan organism.

Developmentally-Regulated Promoter

A developmentally-regulated promoter is active during certaindevelopmental stages or in parts of the plant that undergo developmentalchanges.

Inducible Promoter

An inducible promoter has induced or increased transcription initiationin response to a chemical (for a review see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108), environmental or physicalstimulus, or may be “stress-inducible”, i.e. activated when a plant isexposed to various stress conditions, or a “pathogen-inducible” i.e.activated when a plant is exposed to exposure to various pathogens.

Organ-Specific/Tissue-Specific Promoter

An organ-specific or tissue-specific promoter is one that is capable ofpreferentially initiating transcription in certain organs or tissues,such as the leaves, roots, seed tissue etc. For example, a“root-specific promoter” is a promoter that is transcriptionally activepredominantly in plant roots, substantially to the exclusion of anyother parts of a plant, whilst still allowing for any leaky expressionin these other plant parts. Promoters able to initiate transcription incertain cells only are referred to herein as “cell-specific”.

Examples of root-specific promoters are listed in Table 2b below:

TABLE 2b Examples of root-specific promoters Gene Source Reference RCc3Plant Mol Biol. 1995 January; 27(2): 237-48 Arabidopsis PHT1 Kovama etal., 2005; Mudge et al. (2002, Plant J. 31: 341) Medicago phosphate Xiaoet al., 2006 transporter Arabidopsis Pyk10 Nitz et al. (2001) Plant Sci161 (2): 337-346 root-expressible genes Tingey et al., EMBO J. 6: 1,1987. tobacco auxin- Van der Zaal et al., Plant Mol. Biol. 16, induciblegene 983, 1991. β-tubulin Oppenheimer, et al., Gene 63: 87, 1988.tobacco root- Conkling, et al., Plant Physiol. 93: 1203, 1990. specificgenes B. napus G1-3b gene U.S. Pat. No. 5,401,836 SbPRP1 Suzuki et al.,Plant Mol. Biol. 21: 109-119, 1993. LRX1 Baumberger et al. 2001, Genes &Dev. 15: 1128 BTG-26 Brassica US 20050044585 napus LeAMT1 (tomato)Lauter et al. (1996, PNAS 3: 8139) The LeNRT1-1 Lauter et al. (1996,PNAS 3: 8139) (tomato) class I patatin Liu et al., Plant Mol. Biol. 153:386-395, 1991. gene (potato) KDC1 (Daucus Downey et al. (2000, J. Biol.Chem. 275: 39420) carota) TobRB7 gene W Song (1997) PhD Thesis, NorthCarolina State University, Raleigh, NC USA OsRAB5a (rice) Wang et al.2002, Plant Sci. 163: 273 ALF5 (Arabidopsis) Diener et al. (2001, PlantCell 13: 1625) NRT2; 1Np (N. Quesada et al. (1997, Plant Mol. Biol. 34:265) plumbaginifolia)

A seed-specific promoter is transcriptionally active predominantly inseed tissue, but not necessarily exclusively in seed tissue (in cases ofleaky expression). The seed-specific promoter may be active during seeddevelopment and/or during germination. The seed specific promoter may beendosperm/aleurone/embryo specific. Examples of seed-specific promoters(endosperm/aleurone/embryo specific) are shown in Table 2c to Table 2fbelow. Further examples of seed-specific promoters are given in Qing Quand Takaiwa (Plant Biotechnol. J. 2, 113-125, 2004), which disclosure isincorporated by reference herein as if fully set forth.

TABLE 2c Examples of seed-specific promoters Gene source Referenceseed-specific genes Simon et al., Plant Mol. Biol. 5: 191, 1985;Scofield et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski et al.,Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin Pearson et al., PlantMol. Biol. 18: 235-245, 1992. legumin Ellis et al., Plant Mol. Biol. 10:203-214, 1988. glutelin (rice) Takaiwa et al., Mol. Gen. Genet. 208:15-22, 1986; Takaiwa et al., FEBS Letts. 221: 43-47, 1987. zein Matzkeet al Plant Mol Biol, 14(3): 323-32 1990 napA Stalberg et al, Planta199: 515-519, 1996. wheat LMW and HMW Mol Gen Genet 216: 81-90, 1989;NAR 17: 461-2, 1989 glutenin-1 wheat SPA Albani et al, Plant Cell, 9:171-184, 1997 wheat α, β, γ-gliadins EMBO J. 3: 1409-15, 1984 barleyItr1 promoter Diaz et al. (1995) Mol Gen Genet 248(5): 592-8 barley B1,C, D, hordein Theor Appl Gen 98: 1253-62, 1999; Plant J 4: 343-55, 1993;Mol Gen Genet 250: 750-60, 1996 barley DOF Mena et al, The PlantJournal, 116(1): 53-62, 1998 blz2 EP99106056.7 synthetic promoterVicente-Carbajosa et al., Plant J. 13: 629-640, 1998. rice prolaminNRP33 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 ricea-globulin Glb-1 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998rice OSH1 Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996rice α-globulin REB/OHP-1 Nakase et al. Plant Mol. Biol. 33: 513-522,1997 rice ADP-glucose pyrophos- Trans Res 6: 157-68, 1997 phorylasemaize ESR gene family Plant J 12: 235-46, 1997 sorghum α-kafirin DeRoseet al., Plant Mol. Biol 32: 1029-35, 1996 KNOX Postma-Haarsma et al,Plant Mol. Biol. 39: 257-71, 1999 rice oleosin Wu et al, J. Biochem.123: 386, 1998 sunflower oleosin Cummins et al., Plant Mol. Biol. 19:873-876, 1992 PRO0117, putative rice 40S WO 2004/070039 ribosomalprotein PRO0136, rice alanine unpublished aminotransferase PRO0147,trypsin inhibitor unpublished ITR1 (barley) PRO0151, rice WSI18 WO2004/070039 PRO0175, rice RAB21 WO 2004/070039 PRO005 WO 2004/070039PRO0095 WO 2004/070039 α-amylase (Amy32b) Lanahan et al, Plant Cell 4:203-211, 1992; Skriver et al, Proc Natl Acad Sci USA 88: 7266-7270, 1991cathepsin β-like gene Cejudo et al, Plant Mol Biol 20: 849-856, 1992Barley Ltp2 Kalla et al., Plant J. 6: 849-60, 1994 Chi26 Leah et al.,Plant J. 4: 579-89, 1994 Maize B-Peru Selinger et al., Genetics 149;1125-38, 1998

TABLE 2d examples of endosperm-specific promoters Gene source Referenceglutelin (rice) Takaiwa et al. (1986) Mol Gen Genet 208: 15-22; Takaiwaet al. (1987) FEBS Letts. 221: 43-47 zein Matzke et al., (1990) PlantMol Biol 14(3): 323-32 wheat LMW Colot et al. (1989) Mol Gen Genet 216:81-90, and HMW Anderson et al. (1989) NAR 17: 461-2 glutenin-1 wheat SPAAlbani et al. (1997) Plant Cell 9: 171-184 wheat gliadins Rafalski etal. (1984) EMBO 3: 1409-15 barley Itr1 Diaz et al. (1995) Mol Gen Genet248(5): 592-8 promoter barley B1, C, D, Cho et al. (1999) Theor ApplGenet 98: 1253-62; hordein Muller et al. (1993) Plant J 4: 343-55;Sorenson et al. (1996) Mol Gen Genet 250: 750-60 barley DOF Mena et al,(1998) Plant J 116(1): 53-62 blz2 Onate et al. (1999) J Biol Chem274(14): 9175-82 synthetic promoter Vicente-Carbajosa et al. (1998)Plant J 13: 629-640 rice prolamin Wu et al, (1998) Plant Cell Physiol39(8) 885-889 NRP33 rice globulin Wu et al. (1998) Plant Cell Physiol39(8) 885-889 Glb-1 rice globulin Nakase et al. (1997) Plant Molec Biol33: 513-522 REB/OHP-1 rice ADP-glucose Russell et al. (1997) Trans Res6: 157-68 pyrophosphorylase maize ESR Opsahl-Ferstad et al. (1997) PlantJ 12: 235-46 gene family sorghum kafirin DeRose et al. (1996) Plant MolBiol 32: 1029-35

TABLE 2e Examples of embryo specific promoters: Gene source Referencerice OSH1 Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996KNOX Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999 PRO0151 WO2004/070039 PRO0175 WO 2004/070039 PRO005 WO 2004/070039 PRO0095 WO2004/070039

TABLE 2f Examples of aleurone-specific promoters: Gene source Referenceα-amylase Lanahan et al, Plant Cell 4: 203-211, 1992; (Amy32b) Skriveret al, Proc Natl Acad Sci USA 88: 7266-7270, 1991 cathepsin β-like geneCejudo et al, Plant Mol Biol 20: 849-856, 1992 Barley Ltp2 Kalla et al.,Plant J. 6: 849-60, 1994 Chi26 Leah et al., Plant J. 4: 579-89, 1994Maize B-Peru Selinger et al., Genetics 149; 1125-38, 1998

A green tissue-specific promoter as defined herein is a promoter that istranscriptionally active predominantly in green tissue, substantially tothe exclusion of any other parts of a plant, whilst still allowing forany leaky expression in these other plant parts.

Examples of green tissue-specific promoters which may be used to performthe methods of the invention are shown in Table 2g below.

TABLE 2g Examples of green tissue-specific promoters Gene ExpressionReference Maize Orthophosphate dikinase Leaf specific Fukavama et al.,2001 Maize Phosphoenolpyruvate Leaf specific Kausch et al., 2001carboxylase Rice Phosphoenolpyruvate Leaf specific Liu et al., 2003carboxylase Rice small subunit Rubisco Leaf specific Nomura et al., 2000rice beta expansin EXBP9 Shoot specific WO 2004/070039 Pigeonpea smallsubunit Rubisco Leaf specific Panguluri et al., 2005 Pea RBCS3A Leafspecific

Another example of a tissue-specific promoter is a meristem-specificpromoter, which is transcriptionally active predominantly inmeristematic tissue, substantially to the exclusion of any other partsof a plant, whilst still allowing for any leaky expression in theseother plant parts. Examples of green meristem-specific promoters whichmay be used to perform the methods of the invention are shown in Table2h below.

TABLE 2h Examples of meristem-specific promoters Gene source Expressionpattern Reference rice OSH1 Shoot apical meristem, Sato et al. (1996)from embryo globular stage Proc. Natl. Acad. Sci. to seedling stage USA,93: 8117-8122 Rice metallothionein Meristem specific BAD87835.1 WAK1 &WAK2 Shoot and root apical Wagner & Kohorn meristems, and in (2001)Plant Cell expanding leaves and 13(2): 303-318 sepals

Terminator

The term “terminator” encompasses a control sequence which is a DNAsequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

Modulation

The term “modulation” means in relation to expression or geneexpression, a process in which the expression level is changed by saidgene expression in comparison to the control plant, the expression levelmay be increased or decreased. The original, unmodulated expression maybe of any kind of expression of a structural RNA (rRNA, tRNA) or mRNAwith subsequent translation. The term “modulating the activity” shallmean any change of the expression of the inventive nucleic acidsequences or encoded proteins, which leads to increased yield and/orincreased growth of the plants.

Expression

The term “expression” or “gene expression” means the transcription of aspecific gene or specific genes or specific genetic construct. The term“expression” or “gene expression” in particular means the transcriptionof a gene or genes or genetic construct into structural RNA (rRNA, tRNA)or mRNA with or without subsequent translation of the latter into aprotein. The process includes transcription of DNA and processing of theresulting mRNA product.

Increased Expression/Overexpression

The term “increased expression” or “overexpression” as used herein meansany form of expression that is additional to the original wild-typeexpression level.

Methods for increasing expression of genes or gene products are welldocumented in the art and include, for example, overexpression driven byappropriate promoters, the use of transcription enhancers or translationenhancers. Isolated nucleic acids which serve as promoter or enhancerelements may be introduced in an appropriate position (typicallyupstream) of a non-heterologous form of a polynucleotide so as toupregulate expression of a nucleic acid encoding the polypeptide ofinterest. For example, endogenous promoters may be altered in vivo bymutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.5,565,350; Zarling et al., WO9322443), or isolated promoters may beintroduced into a plant cell in the proper orientation and distance froma gene of the present invention so as to control the expression of thegene.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added may be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence may also be added to the 5′ untranslated region (UTR)or the coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold (Buchman and Berg(1988) Mol. Cell. biol. 8: 4395-4405; Callis et al. (1987) Genes Dev1:1183-1200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofthe maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron areknown in the art. For general information see: The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Endogenous Gene

Reference herein to an “endogenous” gene not only refers to the gene inquestion as found in a plant in its natural form (i.e., without therebeing any human intervention), but also refers to that same gene (or asubstantially homologous nucleic acid/gene) in an isolated formsubsequently (re)introduced into a plant (a transgene). For example, atransgenic plant containing such a transgene may encounter a substantialreduction of the transgene expression and/or substantial reduction ofexpression of the endogenous gene. The isolated gene may be isolatedfrom an organism or may be manmade, for example by chemical synthesis.

Decreased Expression

Reference herein to “decreased expression” or “reduction or substantialelimination” of expression is taken to mean a decrease in endogenousgene expression and/or polypeptide levels and/or polypeptide activityrelative to control plants. The reduction or substantial elimination isin increasing order of preference at least 10%, 20%, 30%, 40% or 50%,60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reducedcompared to that of control plants. Methods for decreasing expressionare known in the art and the skilled person would readily be able toadapt the known methods for silencing so as to achieve reduction ofexpression of an endogenous gene in a whole plant or in parts thereofthrough the use of an appropriate promoter, for example.

For the reduction or substantial elimination of expression an endogenousgene in a plant, a sufficient length of substantially contiguousnucleotides of a nucleic acid sequence is required. In order to performgene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10 or fewer nucleotides, alternatively this may be as much asthe entire gene (including the 5′ and/or 3′ UTR, either in part or inwhole). The stretch of substantially contiguous nucleotides may bederived from the nucleic acid encoding the protein of interest (targetgene), or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest. Preferably, thestretch of substantially contiguous nucleotides is capable of forminghydrogen bonds with the target gene (either sense or antisense strand),more preferably, the stretch of substantially contiguous nucleotideshas, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene(either sense or antisense strand). A nucleic acid sequence encoding a(functional) polypeptide is not a requirement for the various methodsdiscussed herein for the reduction or substantial elimination ofexpression of an endogenous gene.

Examples of various methods for the reduction or substantial eliminationof expression in a plant of an endogenous gene, or for lowering levelsand/or activity of a protein, are known to the skilled in the art. Askilled person would readily be able to adapt the known methods forsilencing, so as to achieve reduction of expression of an endogenousgene in a whole plant or in parts thereof through the use of anappropriate promoter, for example.

This reduction or substantial elimination of expression may be achievedusing routine tools and techniques. A preferred method for the reductionor substantial elimination of endogenous gene expression is byintroducing and expressing in a plant a genetic construct into which thenucleic acid (in this case a stretch of substantially contiguousnucleotides derived from the gene of interest, or from any nucleic acidcapable of encoding an orthologue, paralogue or homologue of any one ofthe protein of interest) is cloned as an inverted repeat (in part orcompletely), separated by a spacer (non-coding DNA).

In such a preferred method, expression of the endogenous gene is reducedor substantially eliminated through RNA-mediated silencing using aninverted repeat of a nucleic acid or a part thereof (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), preferably capableof forming a hairpin structure. The inverted repeat is cloned in anexpression vector comprising control sequences. A non-coding DNA nucleicacid sequence (a spacer, for example a matrix attachment region fragment(MAR), an intron, a polylinker, etc.) is located between the twoinverted nucleic acids forming the inverted repeat. After transcriptionof the inverted repeat, a chimeric RNA with a self-complementarystructure is formed (partial or complete). This double-stranded RNAstructure is referred to as the hairpin RNA (hpRNA). The hpRNA isprocessed by the plant into siRNAs that are incorporated into anRNA-induced silencing complex (RISC). The RISC further cleaves the mRNAtranscripts, thereby substantially reducing the number of mRNAtranscripts to be translated into polypeptides. For further generaldetails see for example, Grierson et al. (1998) WO 98/53083; Waterhouseet al. (1999) WO 99/53050).

Performance of the methods of the invention does not rely on introducingand expressing in a plant a genetic construct into which the nucleicacid is cloned as an inverted repeat, but any one or more of severalwell-known “gene silencing” methods may be used to achieve the sameeffects.

One such method for the reduction of endogenous gene expression isRNA-mediated silencing of gene expression (downregulation). Silencing inthis case is triggered in a plant by a double stranded RNA sequence(dsRNA) that is substantially similar to the target endogenous gene.This dsRNA is further processed by the plant into about 20 to about 26nucleotides called short interfering RNAs (siRNAs). The siRNAs areincorporated into an RNA-induced silencing complex (RISC) that cleavesthe mRNA transcript of the endogenous target gene, thereby substantiallyreducing the number of mRNA transcripts to be translated into apolypeptide. Preferably, the double stranded RNA sequence corresponds toa target gene.

Another example of an RNA silencing method involves the introduction ofnucleic acid sequences or parts thereof (in this case a stretch ofsubstantially contiguous nucleotides derived from the gene of interest,or from any nucleic acid capable of encoding an orthologue, paralogue orhomologue of the protein of interest) in a sense orientation into aplant. “Sense orientation” refers to a DNA sequence that is homologousto an mRNA transcript thereof. Introduced into a plant would thereforebe at least one copy of the nucleic acid sequence. The additionalnucleic acid sequence will reduce expression of the endogenous gene,giving rise to a phenomenon known as co-suppression. The reduction ofgene expression will be more pronounced if several additional copies ofa nucleic acid sequence are introduced into the plant, as there is apositive correlation between high transcript levels and the triggeringof co-suppression.

Another example of an RNA silencing method involves the use of antisensenucleic acid sequences. An “antisense” nucleic acid sequence comprises anucleotide sequence that is complementary to a “sense” nucleic acidsequence encoding a protein, i.e. complementary to the coding strand ofa double-stranded cDNA molecule or complementary to an mRNA transcriptsequence. The antisense nucleic acid sequence is preferablycomplementary to the endogenous gene to be silenced. The complementaritymay be located in the “coding region” and/or in the “non-coding region”of a gene. The term “coding region” refers to a region of the nucleotidesequence comprising codons that are translated into amino acid residues.The term “non-coding region” refers to 5′ and 3′ sequences that flankthe coding region that are transcribed but not translated into aminoacids (also referred to as 5′ and 3′ untranslated regions).

Antisense nucleic acid sequences can be designed according to the rulesof Watson and Crick base pairing. The antisense nucleic acid sequencemay be complementary to the entire nucleic acid sequence (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), but may also be anoligonucleotide that is antisense to only a part of the nucleic acidsequence (including the mRNA 5′ and 3′ UTR). For example, the antisenseoligonucleotide sequence may be complementary to the region surroundingthe translation start site of an mRNA transcript encoding a polypeptide.The length of a suitable antisense oligonucleotide sequence is known inthe art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10nucleotides in length or less. An antisense nucleic acid sequenceaccording to the invention may be constructed using chemical synthesisand enzymatic ligation reactions using methods known in the art. Forexample, an antisense nucleic acid sequence (e.g., an antisenseoligonucleotide sequence) may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acid sequences, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides may be used. Examples of modified nucleotidesthat may be used to generate the antisense nucleic acid sequences arewell known in the art. Known nucleotide modifications includemethylation, cyclization and ‘caps’ and substitution of one or more ofthe naturally occurring nucleotides with an analogue such as inosine.Other modifications of nucleotides are well known in the art.

The antisense nucleic acid sequence can be produced biologically usingan expression vector into which a nucleic acid sequence has beensubcloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Preferably, production of antisense nucleicacid sequences in plants occurs by means of a stably integrated nucleicacid construct comprising a promoter, an operably linked antisenseoligonucleotide, and a terminator.

The nucleic acid molecules used for silencing in the methods of theinvention (whether introduced into a plant or generated in situ)hybridize with or bind to mRNA transcripts and/or genomic DNA encoding apolypeptide to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid sequence which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. Antisense nucleic acid sequences may be introducedinto a plant by transformation or direct injection at a specific tissuesite. Alternatively, antisense nucleic acid sequences can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense nucleic acid sequences can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid sequence to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid sequences canalso be delivered to cells using the vectors described herein.

According to a further aspect, the antisense nucleic acid sequence is ana-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequenceforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual b-units, the strands run parallel to each other(Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisensenucleic acid sequence may also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).

The reduction or substantial elimination of endogenous gene expressionmay also be performed using ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid sequence, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can beused to catalytically cleave mRNA transcripts encoding a polypeptide,thereby substantially reducing the number of mRNA transcripts to betranslated into a polypeptide. A ribozyme having specificity for anucleic acid sequence can be designed (see for example: Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).Alternatively, mRNA transcripts corresponding to a nucleic acid sequencecan be used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules (Bartel and Szostak (1993) Science261, 1411-1418). The use of ribozymes for gene silencing in plants isknown in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al.(1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al.(1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by strategies asdescribed by, among others, Angell and Baulcombe ((1999) Plant J 20(3):357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).

Gene silencing may also occur if there is a mutation on an endogenousgene and/or a mutation on an isolated gene/nucleic acid subsequentlyintroduced into a plant. The reduction or substantial elimination may becaused by a non-functional polypeptide. For example, the polypeptide maybind to various interacting proteins; one or more mutation(s) and/ortruncation(s) may therefore provide for a polypeptide that is still ableto bind interacting proteins (such as receptor proteins) but that cannotexhibit its normal function (such as signalling ligand).

A further approach to gene silencing is by targeting nucleic acidsequences complementary to the regulatory region of the gene (e.g., thepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the gene in target cells. See Helene, C.,Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad.Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.

Other methods, such as the use of antibodies directed to an endogenouspolypeptide for inhibiting its function in planta, or interference inthe signalling pathway in which a polypeptide is involved, will be wellknown to the skilled man. In particular, it can be envisaged thatmanmade molecules may be useful for inhibiting the biological functionof a target polypeptide, or for interfering with the signalling pathwayin which the target polypeptide is involved.

Alternatively, a screening program may be set up to identify in a plantpopulation natural variants of a gene, which variants encodepolypeptides with reduced activity. Such natural variants may also beused for example, to perform homologous recombination.

Artificial and/or natural microRNAs (miRNAs) may be used to knock outgene expression and/or mRNA translation. Endogenous miRNAs are singlestranded small RNAs of typically 19-24 nucleotides long. They functionprimarily to regulate gene expression and/or mRNA translation. Mostplant microRNAs (miRNAs) have perfect or near-perfect complementaritywith their target sequences. However, there are natural targets with upto five mismatches. They are processed from longer non-coding RNAs withcharacteristic fold-back structures by double-strand specific RNases ofthe Dicer family. Upon processing, they are incorporated in theRNA-induced silencing complex (RISC) by binding to its main component,an Argonaute protein. mRNAs serve as the specificity components of RISC,since they base-pair to target nucleic acids, mostly mRNAs, in thecytoplasm. Subsequent regulatory events include target mRNA cleavage anddestruction and/or translational inhibition. Effects of miRNAoverexpression are thus often reflected in decreased mRNA levels oftarget genes.

Artificial microRNAs (amiRNAs), which are typically 21 nucleotides inlength, can be genetically engineered specifically to negativelyregulate gene expression of single or multiple genes of interest.Determinants of plant microRNA target selection are well known in theart. Empirical parameters for target recognition have been defined andcan be used to aid in the design of specific amiRNAs, (Schwab et al.,Dev. Cell 8, 517-527, 2005). Convenient tools for design and generationof amiRNAs and their precursors are also available to the public (Schwabet al., Plant Cell 18, 1121-1133, 2006).

For optimal performance, the gene silencing techniques used for reducingexpression in a plant of an endogenous gene requires the use of nucleicacid sequences from monocotyledonous plants for transformation ofmonocotyledonous plants, and from dicotyledonous plants fortransformation of dicotyledonous plants. Preferably, a nucleic acidsequence from any given plant species is introduced into that samespecies. For example, a nucleic acid sequence from rice is transformedinto a rice plant. However, it is not an absolute requirement that thenucleic acid sequence to be introduced originates from the same plantspecies as the plant in which it will be introduced. It is sufficientthat there is substantial homology between the endogenous target geneand the nucleic acid to be introduced.

Described above are examples of various methods for the reduction orsubstantial elimination of expression in a plant of an endogenous gene.A person skilled in the art would readily be able to adapt theaforementioned methods for silencing so as to achieve reduction ofexpression of an endogenous gene in a whole plant or in parts thereofthrough the use of an appropriate promoter, for example.

Selectable Marker (Gene)/Reporter Gene

“Selectable marker”, “selectable marker gene” or “reporter gene”includes any gene that confers a phenotype on a cell in which it isexpressed to facilitate the identification and/or selection of cellsthat are transfected or transformed with a nucleic acid construct of theinvention. These marker genes enable the identification of a successfultransfer of the nucleic acid molecules via a series of differentprinciples. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptII thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin, or genes conferring resistance to, for example, bleomycin,streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,geneticin (G418), spectinomycin or blasticidin), to herbicides (forexample bar which provides resistance to Basta®; aroA or gox providingresistance against glyphosate, or the genes conferring resistance to,for example, imidazolinone, phosphinothricin or sulfonylurea), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source or xylose isomerase for the utilisation ofxylose, or antinutritive markers such as the resistance to2-deoxyglucose). Expression of visual marker genes results in theformation of colour (for example β-glucuronidase, GUS orβ-galactosidasewith its coloured substrates, for example X-Gal),luminescence (such as the luciferin/luceferase system) or fluorescence(Green Fluorescent Protein, GFP, and derivatives thereof). This listrepresents only a small number of possible markers. The skilled workeris familiar with such markers. Different markers are preferred,depending on the organism and the selection method.

It is known that upon stable or transient integration of nucleic acidsinto plant cells, only a minority of the cells takes up the foreign DNAand, if desired, integrates it into its genome, depending on theexpression vector used and the transfection technique used. To identifyand select these integrants, a gene coding for a selectable marker (suchas the ones described above) is usually introduced into the host cellstogether with the gene of interest. These markers can for example beused in mutants in which these genes are not functional by, for example,deletion by conventional methods. Furthermore, nucleic acid moleculesencoding a selectable marker can be introduced into a host cell on thesame vector that comprises the sequence encoding the polypeptides of theinvention or used in the methods of the invention, or else in a separatevector. Cells which have been stably transfected with the introducednucleic acid can be identified for example by selection (for example,cells which have integrated the selectable marker survive whereas theother cells die). The marker genes may be removed or excised from thetransgenic cell once they are no longer needed. Techniques for markergene removal are known in the art, useful techniques are described abovein the definitions section.

Since the marker genes, particularly genes for resistance to antibioticsand herbicides, are no longer required or are undesired in thetransgenic host cell once the nucleic acids have been introducedsuccessfully, the process according to the invention for introducing thenucleic acids advantageously employs techniques which enable the removalor excision of these marker genes. One such a method is what is known asco-transformation. The co-transformation method employs two vectorssimultaneously for the transformation, one vector bearing the nucleicacid according to the invention and a second bearing the marker gene(s).A large proportion of transformants receives or, in the case of plants,comprises (up to 40% or more of the transformants), both vectors. Incase of transformation with Agrobacteria, the transformants usuallyreceive only a part of the vector, i.e. the sequence flanked by theT-DNA, which usually represents the expression cassette. The markergenes can subsequently be removed from the transformed plant byperforming crosses. In another method, marker genes integrated into atransposon are used for the transformation together with desired nucleicacid (known as the Ac/Ds technology). The transformants can be crossedwith a transposase source or the transformants are transformed with anucleic acid construct conferring expression of a transposase,transiently or stable. In some cases (approx. 10%), the transposon jumpsout of the genome of the host cell once transformation has taken placesuccessfully and is lost. In a further number of cases, the transposonjumps to a different location. In these cases the marker gene must beeliminated by performing crosses. In microbiology, techniques weredeveloped which make possible, or facilitate, the detection of suchevents. A further advantageous method relies on what is known asrecombination systems; whose advantage is that elimination by crossingcan be dispensed with. The best-known system of this type is what isknown as the Cre/lox system. Cre1 is a recombinase that removes thesequences located between the loxP sequences. If the marker gene isintegrated between the loxP sequences, it is removed once transformationhas taken place successfully, by expression of the recombinase. Furtherrecombination systems are the HIN/HIX, FLP/FRT and REP/STB system(Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan etal., J. Cell Biol., 149, 2000: 553-566). A site-specific integrationinto the plant genome of the nucleic acid sequences according to theinvention is possible. Naturally, these methods can also be applied tomicroorganisms such as yeast, fungi or bacteria.

Transgenic/Transgene/Recombinant

For the purposes of the invention, “transgenic”, “transgene” or“recombinant” means with regard to, for example, a nucleic acidsequence, an expression cassette, gene construct or a vector comprisingthe nucleic acid sequence or an organism transformed with the nucleicacid sequences, expression cassettes or vectors according to theinvention, all those constructions brought about by recombinant methodsin which either

-   -   (a) the nucleic acid sequences encoding proteins useful in the        methods of the invention, or    -   (b) genetic control sequence(s) which is operably linked with        the nucleic acid sequence according to the invention, for        example a promoter, or    -   (c) a) and b)        are not located in their natural genetic environment or have        been modified by recombinant methods, it being possible for the        modification to take the form of, for example, a substitution,        addition, deletion, inversion or insertion of one or more        nucleotide residues. The natural genetic environment is        understood as meaning the natural genomic or chromosomal locus        in the original plant or the presence in a genomic library. In        the case of a genomic library, the natural genetic environment        of the nucleic acid sequence is preferably retained, at least in        part. The environment flanks the nucleic acid sequence at least        on one side and has a sequence length of at least 50 bp,        preferably at least 500 bp, especially preferably at least 1000        bp, most preferably at least 5000 bp. A naturally occurring        expression cassette—for example the naturally occurring        combination of the natural promoter of the nucleic acid        sequences with the corresponding nucleic acid sequence encoding        a polypeptide useful in the methods of the present invention, as        defined above—becomes a transgenic expression cassette when this        expression cassette is modified by non-natural, synthetic        (“artificial”) methods such as, for example, mutagenic        treatment. Suitable methods are described, for example, in U.S.        Pat. No. 5,565,350 or WO 00/15815.

A transgenic plant for the purposes of the invention is thus understoodas meaning, as above, that the nucleic acids used in the method of theinvention are not at their natural locus in the genome of said plant, itbeing possible for the nucleic acids to be expressed homologously orheterologously. However, as mentioned, transgenic also means that, whilethe nucleic acids according to the invention or used in the inventivemethod are at their natural position in the genome of a plant, thesequence has been modified with regard to the natural sequence, and/orthat the regulatory sequences of the natural sequences have beenmodified. Transgenic is preferably understood as meaning the expressionof the nucleic acids according to the invention at an unnatural locus inthe genome, i.e. homologous or, preferably, heterologous expression ofthe nucleic acids takes place. Preferred transgenic plants are mentionedherein.

Transformation

The term “introduction” or “transformation” as referred to hereinencompasses the transfer of an exogenous polynucleotide into a hostcell, irrespective of the method used for transfer. Plant tissue capableof subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a genetic construct of thepresent invention and a whole plant regenerated there from. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem). The polynucleotide may be transiently or stablyintroduced into a host cell and may be maintained non-integrated, forexample, as a plasmid. Alternatively, it may be integrated into the hostgenome. The resulting transformed plant cell may then be used toregenerate a transformed plant in a manner known to persons skilled inthe art.

The transfer of foreign genes into the genome of a plant is calledtransformation. Transformation of plant species is now a fairly routinetechnique. Advantageously, any of several transformation methods may beused to introduce the gene of interest into a suitable ancestor cell.The methods described for the transformation and regeneration of plantsfrom plant tissues or plant cells may be utilized for transient or forstable transformation. Transformation methods include the use ofliposomes, electroporation, chemicals that increase free DNA uptake,injection of the DNA directly into the plant, particle gun bombardment,transformation using viruses or pollen and microprojection. Methods maybe selected from the calcium/polyethylene glycol method for protoplasts(Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987)Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plantmaterial (Crossway A et al., (1986) Mol. Gen. Genet. 202: 179-185); DNAor RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327:70) infection with (non-integrative) viruses and the like. Transgenicplants, including transgenic crop plants, are preferably produced viaAgrobacterium-mediated transformation. An advantageous transformationmethod is the transformation in planta. To this end, it is possible, forexample, to allow the agrobacteria to act on plant seeds or to inoculatethe plant meristem with agrobacteria. It has proved particularlyexpedient in accordance with the invention to allow a suspension oftransformed agrobacteria to act on the intact plant or at least on theflower primordia. The plant is subsequently grown on until the seeds ofthe treated plant are obtained (Clough and Bent, Plant J. (1998) 16,735-743). Methods for Agrobacterium-mediated transformation of riceinclude well known methods for rice transformation, such as thosedescribed in any of the following: European patent application EP1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.(Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2):271-282, 1994), which disclosures are incorporated by reference hereinas if fully set forth. In the case of corn transformation, the preferredmethod is as described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), whichdisclosures are incorporated by reference herein as if fully set forth.Said methods are further described by way of example in B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993)128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991) 205-225). The nucleic acids or the construct to be expressed ispreferably cloned into a vector, which is suitable for transformingAgrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. AcidsRes. 12 (1984) 8711). Agrobacteria transformed by such a vector can thenbe used in known manner for the transformation of plants, such as plantsused as a model, like Arabidopsis (Arabidopsis thaliana is within thescope of the present invention not considered as a crop plant), or cropplants such as, by way of example, tobacco plants, for example byimmersing bruised leaves or chopped leaves in an agrobacterial solutionand then culturing them in suitable media. The transformation of plantsby means of Agrobacterium tumefaciens is described, for example, byHöfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is knowninter alia from F. F. White, Vectors for Gene Transfer in Higher Plants;in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D.Kung and R. Wu, Academic Press, 1993, pp. 15-38.

In addition to the transformation of somatic cells, which then have tobe regenerated into intact plants, it is also possible to transform thecells of plant meristems and in particular those cells which developinto gametes. In this case, the transformed gametes follow the naturalplant development, giving rise to transgenic plants. Thus, for example,seeds of Arabidopsis are treated with agrobacteria and seeds areobtained from the developing plants of which a certain proportion istransformed and thus transgenic [Feldman, K A and Marks M D (1987). MolGen Genet. 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and JShell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore,pp. 274-289]. Alternative methods are based on the repeated removal ofthe inflorescences and incubation of the excision site in the center ofthe rosette with transformed agrobacteria, whereby transformed seeds canlikewise be obtained at a later point in time (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, anespecially effective method is the vacuum infiltration method with itsmodifications such as the “floral dip” method. In the case of vacuuminfiltration of Arabidopsis, intact plants under reduced pressure aretreated with an agrobacterial suspension [Bechthold, N (1993). C R AcadSci Paris Life Sci, 316: 1194-1199], while in the case of the “floraldip” method the developing floral tissue is incubated briefly with asurfactant-treated agrobacterial suspension [Clough, S J and Bent AF(1998) The Plant J. 16, 735-743]. A certain proportion of transgenicseeds are harvested in both cases, and these seeds can be distinguishedfrom non-transgenic seeds by growing under the above-described selectiveconditions. In addition the stable transformation of plastids is ofadvantages because plastids are inherited maternally is most cropsreducing or eliminating the risk of transgene flow through pollen. Thetransformation of the chloroplast genome is generally achieved by aprocess which has been schematically displayed in Klaus et al., 2004[Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to betransformed are cloned together with a selectable marker gene betweenflanking sequences homologous to the chloroplast genome. Thesehomologous flanking sequences direct site specific integration into theplastome. Plastidal transformation has been described for many differentplant species and an overview is given in Bock (2001) Transgenicplastids in basic research and plant biotechnology. J Mol. Biol. 2001Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towardscommercialization of plastid transformation technology. TrendsBiotechnol. 21, 20-28. Further biotechnological progress has recentlybeen reported in form of marker free plastid transformants, which can beproduced by a transient co-integrated maker gene (Klaus et al., 2004,Nature Biotechnology 22(2), 225-229).

T-DNA Activation Tagging

T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353),involves insertion of T-DNA, usually containing a promoter (may also bea translation enhancer or an intron), in the genomic region of the geneof interest or 10 kb up- or downstream of the coding region of a gene ina configuration such that the promoter directs expression of thetargeted gene. Typically, regulation of expression of the targeted geneby its natural promoter is disrupted and the gene falls under thecontrol of the newly introduced promoter. The promoter is typicallyembedded in a T-DNA. This T-DNA is randomly inserted into the plantgenome, for example, through Agrobacterium infection and leads tomodified expression of genes near the inserted T-DNA. The resultingtransgenic plants show dominant phenotypes due to modified expression ofgenes close to the introduced promoter.

TILLING

The term “TILLING” is an abbreviation of “Targeted Induced Local LesionsIn Genomes” and refers to a mutagenesis technology useful to generateand/or identify nucleic acids encoding proteins with modified expressionand/or activity. TILLING also allows selection of plants carrying suchmutant variants. These mutant variants may exhibit modified expression,either in strength or in location or in timing (if the mutations affectthe promoter for example). These mutant variants may exhibit higheractivity than that exhibited by the gene in its natural form. TILLINGcombines high-density mutagenesis with high-throughput screeningmethods. The steps typically followed in TILLING are: (a) EMSmutagenesis (Redei G P and Koncz C (1992) In Methods in ArabidopsisResearch, Koncz C, Chua N H, Schell J, eds. Singapore, World ScientificPublishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M,Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) InJ Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol.82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation andpooling of individuals; (c) PCR amplification of a region of interest;(d) denaturation and annealing to allow formation of heteroduplexes; (e)DHPLC, where the presence of a heteroduplex in a pool is detected as anextra peak in the chromatogram; (f) identification of the mutantindividual; and (g) sequencing of the mutant PCR product. Methods forTILLING are well known in the art (McCallum et al., (2000) NatBiotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet. 5(2):145-50).

Homologous Recombination

Homologous recombination allows introduction in a genome of a selectednucleic acid at a defined selected position. Homologous recombination isa standard technology used routinely in biological sciences for lowerorganisms such as yeast or the moss Physcomitrella. Methods forperforming homologous recombination in plants have been described notonly for model plants (Offring a et al. (1990) EMBO J. 9(10): 3077-84)but also for crop plants, for example rice (Terada et al. (2002) NatBiotech 20(10): 1030-4; lida and Terada (2004) Curr Opin Biotech 15(2):132-8), and approaches exist that are generally applicable regardless ofthe target organism (Miller et al, Nature Biotechnol. 25, 778-785,2007).

Yield

The term “yield” in general means a measurable produce of economicvalue, typically related to a specified crop, to an area, and to aperiod of time. Individual plant parts directly contribute to yieldbased on their number, size and/or weight, or the actual yield is theyield per square meter for a crop and year, which is determined bydividing total production (includes both harvested and appraisedproduction) by planted square meters. The term “yield” of a plant mayrelate to vegetative biomass (root and/or shoot biomass), toreproductive organs, and/or to propagules (such as seeds) of that plant.

Early Vigour

“Early vigour” refers to active healthy well-balanced growth especiallyduring early stages of plant growth, and may result from increased plantfitness due to, for example, the plants being better adapted to theirenvironment (i.e. optimizing the use of energy resources andpartitioning between shoot and root). Plants having early vigour alsoshow increased seedling survival and a better establishment of the crop,which often results in highly uniform fields (with the crop growing inuniform manner, i.e. with the majority of plants reaching the variousstages of development at substantially the same time), and often betterand higher yield. Therefore, early vigour may be determined by measuringvarious factors, such as thousand kernel weight, percentage germination,percentage emergence, seedling growth, seedling height, root length,root and shoot biomass and many more.

Increase/Improve/Enhance

The terms “increase”, “improve” or “enhance” are interchangeable andshall mean in the sense of the application at least a 3%, 4%, 5%, 6%,7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%,30%, 35% or 40% more yield and/or growth in comparison to control plantsas defined herein.

Seed Yield

Increased seed yield may manifest itself as one or more of thefollowing: a) an increase in seed biomass (total seed weight) which maybe on an individual seed basis and/or per plant and/or per square meter;b) increased number of flowers per plant; c) increased number of(filled) seeds; d) increased seed filling rate (which is expressed asthe ratio between the number of filled seeds divided by the total numberof seeds); e) increased harvest index, which is expressed as a ratio ofthe yield of harvestable parts, such as seeds, divided by the totalbiomass; and f) increased thousand kernel weight (TKW), and g) increasednumber of primary panicles, which is extrapolated from the number offilled seeds counted and their total weight. An increased TKW may resultfrom an increased seed size and/or seed weight, and may also result froman increase in embryo and/or endosperm size.

An increase in seed yield may also be manifested as an increase in seedsize and/or seed volume. Furthermore, an increase in seed yield may alsomanifest itself as an increase in seed area and/or seed length and/orseed width and/or seed perimeter. Increased seed yield may also resultin modified architecture, or may occur because of modified architecture.

Greenness Index

The “greenness index” as used herein is calculated from digital imagesof plants. For each pixel belonging to the plant object on the image,the ratio of the green value versus the red value (in the RGB model forencoding color) is calculated. The greenness index is expressed as thepercentage of pixels for which the green-to-red ratio exceeds a giventhreshold. Under normal growth conditions, under salt stress growthconditions, and under reduced nutrient availability growth conditions,the greenness index of plants is measured in the last imaging beforeflowering. In contrast, under drought stress growth conditions, thegreenness index of plants is measured in the first imaging afterdrought.

Plant

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,leaves, roots (including tubers), flowers, and tissues and organs,wherein each of the aforementioned comprise the gene/nucleic acid ofinterest. The term “plant” also encompasses plant cells, suspensioncultures, callus tissue, embryos, meristematic regions, gametophytes,sporophytes, pollen and microspores, again wherein each of theaforementioned comprises the gene/nucleic acid of interest.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubsselected from the list comprising Acer spp., Actinidia spp., Abelmoschusspp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp.,Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apiumgraveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avenaspp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasahispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g.Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]),Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa,Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Caryaspp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichoriumendivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp.,Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrumsativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp.,Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpuslongan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g.Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef,Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora,Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica,Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g.Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthusspp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp.,Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp.,Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum,Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzulasylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp.,Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp.,Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp.,Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotianaspp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryzasativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum,Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp.,Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleumpratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp.,Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunusspp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp.,Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubusspp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamumspp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanumintegrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp.,Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao,Trifolium spp., Tripsacum dactyloides, Triticale sp., Triticosecalerimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticumturgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticummonococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus,Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zeamays, Zizania palustris, Ziziphus spp., amongst others.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been found that modulating expression in aplant of a nucleic acid encoding an ASPAT polypeptide gives plantshaving enhanced yield-related traits relative to control plants.According to a first embodiment, the present invention provides a methodfor enhancing yield-related traits in plants relative to control plants,comprising modulating expression in a plant of a nucleic acid encodingan ASPAT polypeptide and optionally selecting for plants having enhancedyield-related traits.

Furthermore surprisingly, it has now been found that increasingexpression in a plant of a nucleic acid sequence encoding an MYB91polypeptide as defined herein, gives plants having increasedyield-related traits relative to control plants. According to a furtherembodiment, the present invention provides a method for increasingyield-related traits in plants relative to control plants, comprisingincreasing expression in a plant of a nucleic acid sequence encoding anMYB91 polypeptide.

Even furthermore surprisingly, it has now been found that modulatingexpression in a plant of a nucleic acid encoding a GASA polypeptidegives plants having enhanced yield-related traits relative to controlplants. According to a further embodiment, the present inventionprovides a method for enhancing yield-related traits in plants relativeto control plants, comprising modulating expression in a plant of anucleic acid encoding a GASA polypeptide.

Yet furthermore surprisingly, it has now been found that modulatingexpression in a plant of a nucleic acid encoding an AUX/IAA polypeptidegives plants having enhanced yield-related traits relative to controlplants. According to a first embodiment, the present invention providesa method for enhancing yield-related traits in plants relative tocontrol plants, comprising modulating expression in a plant of a nucleicacid encoding an AUX/IAA polypeptide and wherein the yield relatedtraits do not encompass increased root growth.

Concerning ASPAT polypeptides, a preferred method for modulating(preferably, increasing) expression of a nucleic acid encoding an ASPATpolypeptide (ASPAT nucleic acid) is by introducing and expressing in aplant a nucleic acid encoding an ASPAT polypeptide. Preferably theincreased expression of the ASPAT nucleic acid and/or the of the ASPATpolypeptide and/or ASPAT activity occurs in one or more subcellularcompartments selected in increasing order of preference from thecytosol, the chloroplast, the peroxisomes, the glyoxisomes and themitochondria of a plant cell.

Cytosolic levels of the ASPAT nucleic acid expression levels and/orASPAT polypeptide and/or ASPAT activity may be increased for example byexpressing an ASPAT nucleic acid encoding a cytosolic isoform.Alternatively, ASPAT nucleic acids encoding isoforms naturally expressedin an organelle of the plant cell may be expressed in the cytosol byremoving the specific organelle targeting motifs. Similarly a naturallyfound cytosolic isoform may be expressed in a preferred organelle byfussing specific acid amino acid motifs encoding known specificsubcellular targeting signals of such organelle. Tools and techniques toexpresses a polypeptide in a preferred organelle of a plant cell arewell known in the art.

Concerning MYB91 polypeptides, a preferred method for increasingexpression in a plant of a nucleic acid sequence encoding an MYB91polypeptide is by introducing and expressing in a plant a nucleic acidsequence encoding an MYB91 polypeptide.

Concerning GASA polypeptides, a preferred method for modulating(preferably, increasing) expression of a nucleic acid encoding a GASApolypeptide is by introducing and expressing in a plant a nucleic acidencoding a GASA polypeptide.

Concerning AUX/IAA polypeptides, a preferred method for modulating(preferably, increasing) expression of a nucleic acid encoding anAUX/IAA polypeptide is by introducing and expressing in a plant anucleic acid encoding an AUX/IAA polypeptide.

Concerning ASPAT polypeptides, any reference hereinafter to a “proteinuseful in the methods of the invention” is taken to mean an ASPATpolypeptide as defined herein. Any reference hereinafter to a “nucleicacid useful in the methods of the invention” is taken to mean a nucleicacid capable of encoding such an ASPAT polypeptide. The nucleic acid tobe introduced into a plant (and therefore useful in performing themethods of the invention) is any nucleic acid encoding the type ofprotein which will now be described, hereafter also named “ASPAT nucleicacid” or “ASPAT gene”.

Concerning MYB91 polypeptides, any reference hereinafter to a “proteinuseful in the methods of the invention” is taken to mean an MYB91polypeptide as defined herein. Any reference hereinafter to a “nucleicacid sequence useful in the methods of the invention” is taken to mean anucleic acid sequence capable of encoding such an MYB91 polypeptide. Thenucleic acid sequence to be introduced into a plant (and thereforeuseful in performing the methods of the invention) is any nucleic acidsequence encoding the type of polypeptide, which will now be described,hereafter also named “MYB91 nucleic acid sequence” or “MYB91 gene”.

Concerning GASA polypeptides, any reference hereinafter to a “proteinuseful in the methods of the invention” is taken to mean a GASApolypeptide as defined herein. Any reference hereinafter to a “nucleicacid useful in the methods of the invention” is taken to mean a nucleicacid capable of encoding such a GASA polypeptide. The nucleic acid to beintroduced into a plant (and therefore useful in performing the methodsof the invention) is any nucleic acid encoding the type of protein whichwill now be described, hereafter also named “GASA nucleic acid” or “GASAgene”.

Concerning AUX/IAA polypeptides, any reference hereinafter to a “protein(or polypeptide) useful in the methods of the invention” is taken tomean an AUX/IAA polypeptide as defined herein. Any reference hereinafterto a “nucleic acid useful in the methods of the invention” is taken tomean a nucleic acid capable of encoding such an AUX/IAA polypeptide. Thenucleic acid to be introduced into a plant (and therefore useful inperforming the methods of the invention) is any nucleic acid encodingthe type of protein which will now be described, hereafter also named“AUX/IAA nucleic acid” or “AUX/IAA gene”.

An “ASPAT polypeptide” as defined herein refers to any polypeptidecomprising an

Aminotransferase, class I and II (Aminotran_(—)1_(—)2) domain (Interproaccession number: IPR004839; pfam accession number: PF00155), andoptionally Aspartate Transaminase activity (EC. 2.6.1.1).

Preferably, an ASPAT polypeptide comprises an Aminotran_(—)1_(—)2 domainhaving in increasing order of preference at least 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to any of theAminotran_(—)1_(—)2 domains as set forth in Tables D1, Table D2 andTable D3.

Preferably the ASPAT polypeptide comprises a motif having at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any one or more of thefollowing motif:

(i) Motif 1 (SEQ ID NO: 207): NPTG; (ii)Motif 2 (SEQ ID NO: 208): IVLLHACAHNPTGVDPT; (iii)Motif 3 (SEQ ID NO: 209): SRLLILCSPSNPTGSVY;wherein any amino acid maybe substituted by a conserved amino acid.

Preferably, the homologue of an ASPAT polypeptide has in increasingorder of preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% overallsequence identity to the amino acid of any of the polypeptides of TableA1, preferably to any of the polypeptides in phylogenetic class 1 ofTable B1, more preferably to SEQ ID NO: 2, even more preferably to SEQID NO: 8, most preferably to SEQ ID NO: 6. In addition the homologue ofan ASPAT protein preferably comprises an Aminotran_(—)1_(—)2 domain asdescribed above. The sequence identity is determined using a globalalignment algorithm, such as the Needleman Wunsch algorithm in theprogram GAP (GCG Wisconsin Package, Accelrys), preferably with defaultparameters and preferably with sequences of mature proteins (i.e.without taking into account secretion signals or transit peptides).Compared to overall sequence identity, the sequence identity willgenerally be higher when only conserved domains or motifs areconsidered.

Alternatively, an ASPAT polypeptide useful in the methods of theinvention has an amino acid sequence which when used in the constructionof a phylogenetic tree, such as the one depicted in FIG. 2 clusters inincreasing order of preference with any of the polypeptides ofphylogenetic class 1, class 2, class 3 and class 4 as set forth in tableB1.

A “MYB91 polypeptide” as defined herein refers to any polypeptidecomprising (i) in increasing order of preference at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to a MYB DNA binding domain with an InterPro accession numberIPR014778, as represented by SEQ ID NO: 269; and (ii) in increasingorder of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or more amino acid sequence identity to a MYB DNAbinding domain with an InterPro accession number IPR014778, asrepresented by SEQ ID NO: 270; and (iii) in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to a Conserved Domain asrepresented by SEQ ID NO: 271.

Alternatively or additionally, a “MYB91 polypeptide” as defined hereinrefers to any polypeptide sequence having in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to a polypeptide asrepresented by SEQ ID NO: 221.

Alternatively or additionally, a “MYB91 polypeptide” as defined hereinrefers to any polypeptide having in increasing order of preference atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or moreamino acid sequence identity to any of the polypeptide sequences givenin Table A2 herein.

Alternatively or additionally, a “MYB91 polypeptide” as defined hereinrefers to any polypeptide sequence which when used in the constructionof a phylogenetic tree of MYB polypeptides, such as the one depicted inFIG. 4, clusters with the MYB91 group of polypeptides rather than withany other group.

A “GASA polypeptide” as defined herein refers to polypeptides comprisingin their native form a secretion signal, the GASA domain PF02704(Interpro IPR003854) and the following three motifs:

Motif 4, (SEQ ID NO: 277) comprising 4 conserved Cys residues: CXXCCXXCX

Wherein X in position 2 can be any amino acid, but preferably one of N,K, M, G, L, I, Q; and wherein X in position 3 can be any amino acid, butpreferably one of V, T, S, M, I, L, H, Y, K; and wherein X in position 6can be any amino acid, but preferably one of Q, A, N, D, L, V, R, H, S,G, K, E, T; and wherein X in position 7 can be any amino acid, butpreferably one of R, T, A, D, K, E, Q, S, W, C; and wherein X inposition 9 can be any amino acid, but preferably one of N, K, R, H, S,G, A, Q, L, D.

Motif 5 (SEQ ID NO: 278): CV(P/L)(P/K/Q/A/S/T)GXX(Q/G/A/S)

Wherein X in position 6 can be any amino acid, but preferably one of T,P, S, Y, V, N, F, L; and wherein X in position 7 can be any amino acid,but preferably one of G, Y, F, S, A, L, V.

Motifs 4 and 5 are adjacent to each other or are separated from eachother by 1 amino acid.

Motif 6 (SEQ ID NO: 279): CY(D/A/T/F/R/N)X(M/L/W/K)

Wherein X in position 4 can be any amino acid, but preferably one of Q,R, S, D, E, N, T, H.

However, the term GASA polypeptide as used in the present invention doesnot encompass GASA4 from Arabidopsis thaliana (SEQ ID NO: 295).

Preferably, the GASA polypeptide useful in the methods of the presentinvention comprises one or more of the following motifs:

Motif 7 (SEQ ID NO: 280): (S/L/Y/K/S/A)C(G/K/M/I/N/L)(L/M/I/V/T/S)CCXXC(N/G/A/K/R/H/S/D)

Wherein X on position 7 can be any amino acid, but preferably one of E,H, G, K, A, Q, S, R, T, N, D, L, V; and wherein X on position 8 can beany amino acid, but preferably one of E, D, K, Q, S, R, A, T, C.

Motif 8 (SEQ ID NO: 281): CVP(T/S/P/A/K/Q)G(S/P/T)(G/Y/L/A/S/F)(S/A/G/Q)(T/S/P/N/D)(R/K/T/Y/L/Q/E)(D/S/H/R/E/N)X(C/I)

Wherein X in position 12 can be any amino acid, but preferably one of E,H, T, A, S, L, V, K, M.

Preferably, motif 7 is immediately followed by motif 8 or is separatedby 1 amino acid from motif 8.

Motif 9 (SEQ ID NO: 282):(P/R/K/T)CY(R/D/T/F/A)(D/Q/R/N/S/T/H/E)(M/K/W/L) (L/V/K/R/T/N/I)

Preferably, motif 8 is immediately followed by motif 9 or is separatedby 1 amino acid from motif 9.

Motif 10 (SEQ ID NO: 283): (K/T)(R/P/V/A)C(L/N/M/I)(F/T)(Y/F/L)C(N/L/Q)(H/Y/K)CC(G/K/E/N/A/R)(W/R/K/T/S/A)C(Q/L/R)CV(P/L)(P/S/K/A)G(Y/T/V/N/F/L)(V/Y/F)G Motif 11 (SEQ ID NO: 284):(N/H)K(G/D/E/Q/A)(C/E/T/S/F/A/V)(C/W)(S/P)CY(N/R)(N/D)(W/L/M)(K/T/E)(T/K/E/N)(Q/K) Motif 12 (SEQ ID NO: 285)(N/R)(G/C)(S/K)(H/Q/A/N/K/G)(K/T)(G/S/Q/A/K) (H/Y/F)(K/T/R/H)

Alternatively, the homologue of a GASA protein has in increasing orderof preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identityto the amino acid represented by SEQ ID NO: 276, provided that thehomologous protein comprises the conserved motifs as outlined above. Theoverall sequence identity is determined using a global alignmentalgorithm, such as the Needleman Wunsch algorithm in the program GAP(GCG Wisconsin Package, Accelrys), preferably with default parametersand preferably with sequences of mature proteins (i.e. without takinginto account secretion signals or transit peptides). Compared to overallsequence identity, the sequence identity will generally be higher whenonly conserved domains or motifs are considered.

Preferably, the polypeptide sequence which when used in the constructionof a phylogenetic tree, such as the one depicted in FIG. 9, clusterswith the group of GASA polypeptides comprising the amino acid sequencerepresented by SEQ ID NO: 276 (or SEQ ID NO: 291 or SEQ ID NO: 292)rather than with any other group. It should be noted that GASA4 fromArabidopsis thaliana (SEQ ID NO: 295) is excluded from the group of GASAproteins as defined in the present invention.

An “AUX/IAA polypeptide” as defined herein refers to any polypeptidecomprising an AUX/IAA domain (PFAM accession number PF02309, InterProentry IPR003311). An “AUX/IAA polypeptide” as defined herein does notcomprise the motif represented by SEQ ID NO: 670:(K/N)(I/M/L)F(S/Y)(Q/G)L (IAA2 motif).

AUX/IAA polypeptides of the invention have equivalent amino acidstructure and function as the AUX/IAA family of transcription factorsand homologues thereof.

The structure and function of AUX/IAA domains are well known in the art.Typically they can be found in AUX/IAA transcription factors of plants.Members of the AUX/IAA family of transcription factors from plant originare well known in the art. A compilation of AUX/IAA polypeptides asfound in the viridiplantae kingdom can be found in dedicated databasessuch as the so called “plant transcription database (PInTFDB)”maintained by the university of Postdam (Germany) and described byRiano-Pacho et al. BMC Bioinformatics 2007 8:47.

In the PInTFDB database the members of the AUX/IAA family are identifiedas polypeptides having a AUX/IAA domain (PFAM accession number: PF02309)and not having an Auxin_resp domain (pfam accession number: PF06507);Auxin_resp domains are typically found in ARF polypeptides and typicallyabsent from AUX/IAA polypeptides.

An Example of an AUX/IAA domain as found between amino acid coordinates5-171 of SEQ ID NO: 432. AUX/IAA domains having sequence similarity tothe domain as present in SEQ ID NO: 432 are present in the polypeptidesof Table A4.

In a one embodiment of the invention, to perform the methods of theinvention there is provided a preferred an AUX/IAA polypeptide, alsoreferred to as IAA14-like polypeptide, which comprises an AUX/IAA domainhaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the amino acid of the AUX/IAA domain represented bythe amino acids 1 to 220 in SEQ ID NO: 738 (FIG. 13).

Preferably the IAA14-like polypeptide comprises at least one, and inincreasing order of preference, 2, 3, 4, 5, or all six of the followingmotifs:

Motif 13, SEQ ID NO: 739: (K/R/E/D)(A/E/D)TEL(C/R)LG(L/I)(P/G)Motif 14, SEQ ID NO: 740: KRGF(S/A)ET Motif 15, SEQ ID NO: 741:VGWPP(V/I)R Motif 16, SEQ ID NO: 742: GAPYLRK(V/I)DLXX(Y/F)wherein X on position 11 can be any amino acid, preferably X on position11 is one of K, T, R, N, S, or Q and wherein X on position 12 can be anyamino acid, preferably X on position 12 is one of N, L, T, N, V, I, orC.

Motif 17, SEQ ID NO: 743: (S/N/G)(S/W/T)(E/D/G)(Y/F/H)(V/A/E)(P/L/V/I)(S/T/A)YEDKD(N/G)D(W/L)M(L/F)(V/I)GDVP Motif 18, SEQ ID NO: 744:(S/T)C(K/R/Q)(R/K)(L/I)R(I/L)(M/I)K(G/S/E)(S/K/T) (E/D)(A/T)Preferably motif 15 is: VGWPPVR Motif 16 is preferably:GAPYLRK(V/I)DL(K/T/R/N)(M/L)Y Motif 17 is preferably:(S/N/G)(S/W/T)(E/D)YVP(S/T)YEDKDNDWM(L/F)VGDVP Motif 18 is preferably:(S/T)CK(R/K)(L/I)R(I/L)MK(G/S)(S/K/T)EA

Preferably the AUX/IAA polypeptide of the invention has in increasingorder of preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the amino acid of an AUX/IAA domain, preferably to theAUX/IAA domain of any of the polypeptides of Table A4, most preferablyto the AUX/IAA domain of SEQ ID NO: 432 as represented by the aminoacids located between amino acid coordinates 5 to 171.

Preferably, the IAA14-like polypeptide sequence which when used in theconstruction of a phylogenetic tree, as depicted in FIG. 1 in Remingtonet al. (Plant Physiol. 135, 1738-1752, 2004), clusters with group A ofthe IAA14-like polypeptides, which comprises the amino acid sequencerepresented by SEQ ID NO: 738, rather than with any other group (seealso FIG. 15).

Alternatively, the homologue of an AUX/IAA protein has in increasingorder of preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overallsequence identity to the amino acid represented by any of thepolypeptides of Table A4 or Table A5 preferably by SEQ ID NO: 432 or SEQID NO: 738, provided that the homologous protein comprises one or moreof the conserved motifs as outlined above. The overall sequence identityis determined using a global alignment algorithm, such as the NeedlemanWunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys),preferably with default parameters and preferably with sequences ofmature proteins (i.e. without taking into account secretion signals ortransit peptides). Compared to overall sequence identity, the sequenceidentity will generally be higher when only conserved domains or motifsare considered.

In a preferred embodiment, the polypeptide sequence which when used inthe construction of a phylogenetic tree, as depicted in FIG. 1 inRemington et al. (Plant Physiol. 135, 1738-1752, 2004), clusters withgroup A of the IAA14-like polypeptides, which comprises the amino acidsequence represented by SEQ ID NO: 738, rather than with any other group(see also FIG. 15).

The terms “domain”, “signature” and “motif” are defined in the“definitions” section herein. Specialist databases exist for theidentification of domains, for example, SMART (Schultz et al. (1998)Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) NucleicAcids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids.Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalizedprofile syntax for biomolecular sequences motifs and its function inautomatic sequence interpretation. (In) ISMB-94; Proceedings 2ndInternational Conference on Intelligent Systems for Molecular Biology.Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61,AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137,(2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280(2002)). A set of tools for in silico analysis of protein sequences isavailable on the ExPASy proteomics server (Swiss Institute ofBioinformatics (Gasteiger et al., ExPASy: the proteomics server forin-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs may also be identified using routinetechniques, such as by sequence alignment.

Concerning MYB91 polypeptides, an alignment of the polypeptides of TableA2 herein, is shown in FIG. 5. Such alignments are useful foridentifying the most conserved domains or motifs between the MYB91polypeptides as defined herein. Examples of such domains are (i) a MYBDNA binding domain with an InterPro accession number IPR014778, asrepresented by SEQ ID NO: 269 and/or by SEQ ID NO: 270 (marked by X's inFIG. 5); and (ii) a MYB DNA transcription factor with an InterPro entryIPR015495 (also marked by X's in FIG. 2). Another such domain is aC-terminal Conserved Domain as represented by SEQ ID NO: 271, alsomarked by X's in FIG. 5.

Methods for the alignment of sequences for comparison are well known inthe art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAPuses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find the global (i.e. spanning the complete sequences)alignment of two sequences that maximizes the number of matches andminimizes the number of gaps. The BLAST algorithm (Altschul et al.(1990) J Mol Biol 215: 403-10) calculates percent sequence identity andperforms a statistical analysis of the similarity between the twosequences. The software for performing BLAST analysis is publiclyavailable through the National Centre for Biotechnology Information(NCBI). Homologues may readily be identified using, for example, theClustalW multiple sequence alignment algorithm (version 1.83), with thedefault pairwise alignment parameters, and a scoring method inpercentage. Global percentages of similarity and identity may also bedetermined using one of the methods available in the MatGAT softwarepackage (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29.MatGAT: an application that generates similarity/identity matrices usingprotein or DNA sequences.). Minor manual editing may be performed tooptimise alignment between conserved motifs, as would be apparent to aperson skilled in the art. Furthermore, instead of using full-lengthsequences for the identification of homologues, specific domains mayalso be used. The sequence identity values may be determined over theentire nucleic acid or amino acid sequence or over selected domains orconserved motif(s), using the programs mentioned above using the defaultparameters. For local alignments, the Smith-Waterman algorithm isparticularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol.147(1); 195-7).

Concerning MYB91 polypeptides, example 3 herein describes in Table B thepercentage identity between the MYB91 polypeptide as represented by SEQID NO: 221 and the MYB91 polypeptides listed in Table A2, which can beas low as 52% amino acid sequence identity. In some instances, thedefault parameters may be adjusted to modify the stringency of thesearch. For example using BLAST, the statistical significance threshold(called “expect” value) for reporting matches against database sequencesmay be increased to show less stringent matches. This way, short nearlyexact matches may be identified.

Concerning GASA polypeptides, an alignment can for example be made fromthe mature protein sequences, that is, without secretion signal peptide.Methods for identifying signal peptides are well known in the art, seefor example Bendtsen et al., J. Mol. Biol., 340:783-795 (2004).

The task of protein subcellular localisation prediction is important andwell studied. Knowing a protein's localisation helps elucidate itsfunction. Experimental methods for protein localization range fromimmunolocalization to tagging of proteins using green fluorescentprotein (GFP) or beta-glucuronidase (GUS). Such methods are accuratealthough labor-intensive compared with computational methods. Recentlymuch progress has been made in computational prediction of proteinlocalisation from sequence data. Among algorithms well known to a personskilled in the art are available at the ExPASy Proteomics tools hostedby the Swiss Institute for Bioinformatics, for example, PSort, TargetP,ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM,and others. By applying the PSort algorithm to an MYB91 polypeptide asrepresented by SEQ ID NO: 221, a predicted nuclear subcellularlocalisation is obtained.

Furthermore, ASPAT polypeptides typically have Aspartate Transaminasealso called Aspartate Transferase activity. Tools and techniques formeasuring Aspartate Transaminase activity are well known in the art.Aspartate Transaminase activity may be for example assayed in vivo bycomplementation of E. coli strains defective in the activity asdescribed by De la Torre et al. 2006. Alternatively, a biochemicaldetermination of Aspartate Transferase activity may be carried out asfor example described in De la Torre et al. 2006.

In addition, ASPAT polypeptides, when expressed in rice according to themethods of the present invention as outlined in the Examples section,give plants having increased yield related traits, in particularincreased seed yield.

GASA polypeptides, when expressed in rice according to the methods ofthe present invention as outlined in the examples section, give plantshaving increased yield related traits, in particular increased totalweight of seeds and/or increased number of filled seeds, and/orincreased harvest index.

Furthermore, transgenic plants expressing GASA polypeptides (at least intheir native form) may have enhanced tolerance to heat stress (Ko et al,2007). Tools and techniques for measuring resistance of plants to heatstress are well known in the art, see for example the methods describedin Ko et al., 2007.

Furthermore, AUX/IAA polypeptides (at least in their native form)typically have protein binding activity: AUX/IAA polypeptides bind toARF (Auxin Response Factor) polypeptides. Tools and techniques formeasuring protein binding activity are well known in the art and includefor example, immuno precipitation of protein complexes and yeast twohybrid. Tools and techniques for measuring the association of AUX/IAAand ARF polypeptide are well known in the art., and include for exampleyeast two hybrid analysis (see for example Fukaki et al. (Plant J. 44,382-395, 2005).

Typically AUX/IAA polypeptides of the invention comprise an EAR domain(Ohata et al; Plant Cell. 2001 13(8):1959-68), which is a well knownprotein domain that typically confers repression activity to thetranscription factors that comprising such domain. The AUX/IAApolypeptides of the invention have preferably transcription repressionactivity.

Concerning IAA14-like polypeptides, they (at least in their native form)typically associate with ARF7 or ARF19 proteins. Tools and techniquesfor measuring this association are well known in the art., and includefor example yeast two hybrid analysis (see for example Fukaki et al.(Plant J. 44, 382-395, 2005) Further details are provided in theExamples section.

In addition, AUX/IAA polypeptides, when expressed in rice according tothe methods of the present invention as outlined in the Examplessection, give plants having increased yield related traits selected formincreased harvest index, increased root biomass, increased green biomassand increased seed yield.

In addition, AUX/IAA polypeptides, when expressed in rice according tothe methods of the present invention as outlined in the Examplessection, give plants having increased yield related traits such asincrease seed fill rate and increased harvest index.

In addition, IAA14-like polypeptides, when expressed in rice accordingto the methods of the present invention as outlined in the Examplessection, give plants having increased yield related traits, preferablyincreased seed yield.

Additionally, AUX/IAA polypeptides may display a preferred subcellularlocalization, typically one or more of nuclear, citoplasmic,chloroplastic, or mitochondrial. The task of protein subcellularlocalisation prediction is important and well studied. Knowing aprotein's localisation helps elucidate its function. Experimentalmethods for protein localization range from immunolocalization totagging of proteins using green fluorescent protein (GFP) orbeta-glucuronidase (GUS). Such methods are accurate althoughlabor-intensive compared with computational methods. Recently muchprogress has been made in computational prediction of proteinlocalisation from sequence data. Among algorithms well known to a personskilled in the art are available at the ExPASy Proteomics tools hostedby the Swiss Institute for Bioinformatics, for example, PSort, TargetP,ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM,and others.

Concerning ASPAT polypeptides, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 1, encoding the polypeptide sequence of SEQ ID NO: 2. However,performance of the invention is not restricted to these sequences; themethods of the invention may advantageously be performed using anyASPAT-encoding nucleic acid or ASPAT polypeptide as defined herein.

Examples of nucleic acids encoding ASPAT polypeptides are given in TableA1 of The Examples section herein. Such nucleic acids are useful inperforming the methods of the invention. The amino acid sequences givenin Table A1 of The Examples section are example sequences of orthologuesand paralogues of the ASPAT polypeptide represented by SEQ ID NO: 2, theterms “orthologues” and “paralogues” being as defined herein. Furtherorthologues and paralogues may readily be identified by performing aso-called reciprocal blast search. Typically, this involves a firstBLAST involving BLASTing a query sequence (for example using any of thesequences listed in Table A1 of The Examples section) against anysequence database, such as the publicly available NCBI database. BLASTNor TBLASTX (using standard default values) are generally used whenstarting from a nucleotide sequence, and BLASTP or TBLASTN (usingstandard default values) when starting from a protein sequence. TheBLAST results may optionally be filtered. The full-length sequences ofeither the filtered results or non-filtered results are then BLASTedback (second BLAST) against sequences from the organism from which thequery sequence is derived (where the query sequence is SEQ ID NO: 1 orSEQ ID NO: 2, the second BLAST would therefore be against ricesequences). The results of the first and second BLASTs are thencompared. A paralogue is identified if a high-ranking hit from the firstblast is from the same species as from which the query sequence isderived, a BLAST back then ideally results in the query sequence amongstthe highest hits; an orthologue is identified if a high-ranking hit inthe first BLAST is not from the same species as from which the querysequence is derived, and preferably results upon BLAST back in the querysequence being among the highest hits.

Concerning MYB91 polypeptides, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 220, encoding the MYB91 polypeptide sequence of SEQ ID NO: 221.However, performance of the invention is not restricted to thesesequences; the methods of the invention may advantageously be performedusing any nucleic acid sequence encoding an MYB91 polypeptide as definedherein.

Examples of nucleic acid sequences encoding MYB91 polypeptides are givenin Table A2 of Example 1 herein. Such nucleic acid sequences are usefulin performing the methods of the invention. The polypeptide sequencesgiven in Table A2 of Example 1 are example sequences of orthologues andparalogues of the MYB91 polypeptide represented by SEQ ID NO: 221, theterms “orthologues” and “paralogues” being as defined herein. Furtherorthologues and paralogues may readily be identified by performing aso-called reciprocal blast search. Typically, this involves a firstBLAST involving BLASTing a query sequence (for example using any of thesequences listed in Table A1 of Example 1) against any sequencedatabase, such as the publicly available NCBI database. BLASTN orTBLASTX (using standard default values) are generally used when startingfrom a nucleotide sequence, and BLASTP or TBLASTN (using standarddefault values) when starting from a protein sequence. The BLAST resultsmay optionally be filtered. The full-length sequences of either thefiltered results or non-filtered results are then BLASTed back (secondBLAST) against sequences from the organism from which the query sequenceis derived (where the query sequence is SEQ ID NO: 220 or SEQ ID NO:221, the second BLAST would therefore be against Populus trichocarpasequences). The results of the first and second BLASTs are thencompared. A paralogue is identified if a high-ranking hit from the firstblast is from the same species as from which the query sequence isderived, a BLAST back then ideally results in the query sequence amongstthe highest hits; an orthologue is identified if a high-ranking hit inthe first BLAST is not from the same species as from which the querysequence is derived, and preferably results upon BLAST back in the querysequence being among the highest hits.

Concerning GASA polypeptides, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 275, encoding the polypeptide sequence of SEQ ID NO: 276; and withSEQ ID NO: 361, encoding SEQ ID NO: 291. However, performance of theinvention is not restricted to these sequences; the methods of theinvention may advantageously be performed using any GASA-encodingnucleic acid or GASA polypeptide as defined herein. In a preferredembodiment, the nucleic acid encoding the GASA polypeptide, whenexpressed in a plant, is a heterologous nucleic acid, the heterologousnucleic acid being sufficiently different from the endogenous GASAnucleic acid such that gene silencing is avoided.

Examples of nucleic acids encoding GASA polypeptides are given in TableA3 of the Examples section herein. Such nucleic acids are useful inperforming the methods of the invention. The amino acid sequences givenin Table A3 of the Examples section are example sequences of orthologuesand paralogues of the GASA polypeptide represented by SEQ ID NO: 276,the terms “orthologues” and “paralogues” being as defined herein.Further orthologues and paralogues may readily be identified byperforming a so-called reciprocal blast search. Typically, this involvesa first BLAST involving BLASTing a query sequence (for example using anyof the sequences listed in Table A3 of the Examples section) against anysequence database, such as the publicly available NCBI database. BLASTNor TBLASTX (using standard default values) are generally used whenstarting from a nucleotide sequence, and BLASTP or TBLASTN (usingstandard default values) when starting from a protein sequence. TheBLAST results may optionally be filtered. The full-length sequences ofeither the filtered results or non-filtered results are then BLASTedback (second BLAST) against sequences from the organism from which thequery sequence is derived (where the query sequence is SEQ ID NO: 275 orSEQ ID NO: 276, the second BLAST would therefore be against tomato(Solanum lycopersicum) sequences; where the query sequence is SEQ ID NO:361 or SEQ ID NO: 291, the second BLAST would therefore be againstpoplar sequences). The results of the first and second BLASTs are thencompared. A paralogue is identified if a high-ranking hit from the firstblast is from the same species as from which the query sequence isderived, a BLAST back then ideally results in the query sequence amongstthe highest hits; an orthologue is identified if a high-ranking hit inthe first BLAST is not from the same species as from which the querysequence is derived, and preferably results upon BLAST back in the querysequence being among the highest hits.

Concerning AUX/IAA polypeptides, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 431 or by SEQ ID NO: 737, encoding the polypeptide sequence of SEQID NO: 432 or by SEQ ID NO: 738. However, performance of the inventionis not restricted to these sequences; the methods of the invention mayadvantageously be performed using any AUX/IAA-encoding nucleic acid orIAA14-like polypeptide as defined herein.

Examples of nucleic acids encoding AUX/IAA polypeptides are given inTable A4 and in Table A5 of the Examples section herein. Such nucleicacids are useful in performing the methods of the invention. The aminoacid sequences given in Table A4 and in Table A5 of the Examples sectionare example sequences of orthologues and paralogues of the AUX/IAApolypeptide represented by SEQ ID NO: 432 or by SEQ ID NO: 738, theterms “orthologues” and “paralogues” being as defined herein. Furtherorthologues and paralogues may readily be identified by performing aso-called reciprocal blast search. Typically, this involves a firstBLAST involving BLASTing a query sequence (for example using any of thesequences listed in Table A4 or Table A5 of the Examples section)against any sequence database, such as the publicly available NCBIdatabase. BLASTN or TBLASTX (using standard default values) aregenerally used when starting from a nucleotide sequence, and BLASTP orTBLASTN (using standard default values) when starting from a proteinsequence. The BLAST results may optionally be filtered. The full-lengthsequences of either the filtered results or non-filtered results arethen BLASTed back (second BLAST) against sequences from the organismfrom which the query sequence is derived (where the query sequence isSEQ ID NO: 431 or SEQ ID NO: 432, the second BLAST would therefore beagainst Arabidopsis sequences). The results of the first and secondBLASTs are then compared. A paralogue is identified if a high-rankinghit from the first blast is from the same species as from which thequery sequence is derived, a BLAST back then ideally results in thequery sequence amongst the highest hits; an orthologue is identified ifa high-ranking hit in the first BLAST is not from the same species asfrom which the query sequence is derived, and preferably results uponBLAST back in the query sequence being among the highest hits.

High-ranking hits are those having a low E-value. The lower the E-value,the more significant the score (or in other words the lower the chancethat the hit was found by chance). Computation of the E-value is wellknown in the art. In addition to E-values, comparisons are also scoredby percentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In the case oflarge families, ClustalW may be used, followed by a neighbour joiningtree, to help visualize clustering of related genes and to identifyorthologues and paralogues.

Nucleic acid variants may also be useful in practising the methods ofthe invention. Examples of such variants include nucleic acids encodinghomologues and derivatives of any one of the amino acid sequences givenin Table A1 to A5 of The Examples section, the terms “homologue” and“derivative” being as defined herein. Also useful in the methods of theinvention are nucleic acids encoding homologues and derivatives oforthologues or paralogues of any one of the amino acid sequences givenin Table A1 to A5 of The Examples section. Homologues and derivativesuseful in the methods of the present invention have substantially thesame biological and functional activity as the unmodified protein fromwhich they are derived. Also included are nucleic acids variants inwhich codon usage is optimised or in which miRNA target sites areremoved.

Further nucleic acid variants useful in practising the methods of theinvention include portions of nucleic acids encoding ASPAT polypeptides,or MYB91 polypeptides, or GASA polypeptides, or AUX/IAA polypeptides,nucleic acids hybridising to nucleic acids encoding ASPAT polypeptides,or MYB91 polypeptides, or GASA polypeptides, or AUX/IAA polypeptides,splice variants of nucleic acids encoding ASPAT polypeptides, or MYB91polypeptides, or GASA polypeptides, or AUX/IAA polypeptides, allelicvariants of nucleic acids encoding ASPAT polypeptides, or MYB91polypeptides, or GASA polypeptides, or AUX/IAA polypeptides, andvariants of nucleic acids encoding ASPAT polypeptides, or MYB91polypeptides, or GASA polypeptides, or AUX/IAA polypeptides, obtained bygene shuffling. The terms hybridising sequence, splice variant, allelicvariant and gene shuffling are as described herein.

Nucleic acids encoding ASPAT polypeptides, or MYB91 polypeptides, orGASA polypeptides, or AUX/IAA polypeptides, need not be full-lengthnucleic acids, since performance of the methods of the invention doesnot rely on the use of full-length nucleic acid sequences. According tothe present invention, there is provided a method for enhancingyield-related traits in plants, comprising introducing and expressing ina plant a portion of any one of the nucleic acid sequences given inTable A1 to A5 of The Examples section, or a portion of a nucleic acidencoding an orthologue, paralogue or homologue of any of the amino acidsequences given in Table A1 to A5 of The Examples section.

A portion of a nucleic acid may be prepared, for example, by making oneor more deletions to the nucleic acid. The portions may be used inisolated form or they may be fused to other coding (or non-coding)sequences in order to, for example, produce a protein that combinesseveral activities. When fused to other coding sequences, the resultantpolypeptide produced upon translation may be bigger than that predictedfor the protein portion.

Concerning ASPAT polypeptides, portions useful in the methods of theinvention, encode an ASPAT polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesgiven in Table A1 of The Examples section. Preferably, the portion is aportion of any one of the nucleic acids given in Table A1 of TheExamples section, or is a portion of a nucleic acid encoding anorthologue or paralogue of any one of the amino acid sequences given inTable A1 of The Examples section.

Preferably the portion is at least 100, 200, 300, 400, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000 consecutive nucleotides inlength, the consecutive nucleotides being of any one of the nucleic acidsequences given in Table A1 of The Examples section, or of a nucleicacid encoding an orthologue or paralogue of any one of the amino acidsequences given in Table A1 of The Examples section. Even morepreferably the portion is a portion of the nucleic acid of SEQ ID NO: 1,most preferably is the nucleic acid of SEQ ID NO: 3. Preferably, theportion encodes a fragment of an amino acid sequence which, when used inthe construction of a phylogenetic tree, such as the one depicted inFIG. 2 clusters in increasing order of preference with any of thepolypeptides in phylogenetic class 1, class 2, class 3 and class 4 asset forth in Table B1. Most preferably the portion encodes the aminoacid fragment as represented by SEQ ID NO: 4.

Concerning MYB91 polypeptides, portions useful in the methods of theinvention, encode an MYB91 polypeptide as defined herein, and havesubstantially the same biological activity as the polypeptide sequencesgiven in Table A2 of Example 1. Preferably, the portion is a portion ofany one of the nucleic acid sequences given in Table A2 of Example 1, oris a portion of a nucleic acid sequence encoding an orthologue orparalogue of any one of the polypeptide sequences given in Table A2 ofExample 1. Preferably the portion is, in increasing order of preferenceat least 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 ormore consecutive nucleotides in length, the consecutive nucleotidesbeing of any one of the nucleic acid sequences given in Table A2 ofExample 1, or of a nucleic acid sequence encoding an orthologue orparalogue of any one of the polypeptide sequences given in Table A2 ofExample 1. Preferably, the portion is a portion of a nucleic sequenceencoding a polypeptide sequence comprising (i) in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to a MYB DNA bindingdomain with an InterPro accession number IPR014778, as represented bySEQ ID NO: 269; and (ii) in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acidsequence identity to a MYB DNA binding domain with an InterPro accessionnumber IPR014778, as represented by SEQ ID NO: 270; and (iii) inincreasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to aConserved Domain as represented by SEQ ID NO: 271. More preferably, theportion is a portion of a nucleic sequence encoding a polypeptidesequence having in increasing order of preference at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acidsequence identity to the MYB91 polypeptide as represented by SEQ ID NO:221 or to any of the polypeptide sequences given in Table A2 herein.Most preferably, the portion is a portion of the nucleic acid sequenceof SEQ ID NO: 220.

Concerning GASA polypeptides, portions useful in the methods of theinvention, encode a GASA polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesgiven in Table A3 of the Examples section. Preferably, the portion is aportion of any one of the nucleic acids given in Table A3 of theExamples section, or is a portion of a nucleic acid encoding anorthologue or paralogue of any one of the amino acid sequences given inTable A3 of the Examples section. Preferably the portion is at least200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000consecutive nucleotides in length, the consecutive nucleotides being ofany one of the nucleic acid sequences given in Table A3 of the Examplessection, or of a nucleic acid encoding an orthologue or paralogue of anyone of the amino acid sequences given in Table A3 of the Examplessection. Most preferably the portion is a portion of the nucleic acid ofSEQ ID NO: 275. Preferably, the portion encodes a fragment of an aminoacid sequence which, when used in the construction of a phylogenetictree, such as the one depicted in FIG. 9, clusters with the group ofGASA polypeptides comprising the amino acid sequence represented by SEQID NO: 276 (or SEQ ID NO: 291 or SEQ ID NO: 292) rather than with anyother group.

Concerning AUX/IAA polypeptides, portions useful in the methods of theinvention, encode an AUX/IAA polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesgiven in Table A4 or in Table A5 of the Examples section. Preferably,the portion is a portion of any one of the nucleic acids given in TableA4 or in Table A5 of the Examples section, or is a portion of a nucleicacid encoding an orthologue or paralogue of any one of the amino acidsequences given in Table A4 or in Table A5 of the Examples section.Preferably the portion is at least 100, 200, 300, 400, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000 consecutive nucleotides inlength, the consecutive nucleotides being of any one of the nucleic acidsequences given in Table A4 or in Table A5 of the Examples section, orof a nucleic acid encoding an orthologue or paralogue of any one of theamino acid sequences given in Table A4 or in Table A5 of the Examplessection. Most preferably the portion is a portion of the nucleic acid ofSEQ ID NO: 431 or of SEQ ID NO: 737. Preferably, the portion encodes afragment of an amino acid sequence comprising an AUX/IAA domain (PFAMaccession number PF2309, InterPro entry IPR003311).

In the case of an IAA14-like polypeptide, preferably, the portionencodes a fragment of an amino acid sequence which, when used in theconstruction of a phylogenetic tree, as depicted in FIG. 1 in Remingtonet al. (Plant Physiol. 135, 1738-1752, 2004), clusters with group A ofthe IAA14-like polypeptides, which comprises the amino acid sequencerepresented by SEQ ID NO: 738, rather than with any other group (seealso FIG. 13).

Another nucleic acid variant useful in the methods of the invention is anucleic acid capable of hybridising, under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding an ASPAT polypeptide, or an MYB91 polypeptide, or a GASApolypeptide, or an AUX/IAA polypeptide, as defined herein, or with aportion as defined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a nucleic acid capable of hybridizing to any oneof the nucleic acids given in Table A1 to A5 of The Examples section, orcomprising introducing and expressing in a plant a nucleic acid capableof hybridising to a nucleic acid encoding an orthologue, paralogue orhomologue of any of the nucleic acid sequences given in Table A1 to A5of The Examples section.

Concerning ASPAT polypeptides, hybridising sequences useful in themethods of the invention encode an ASPAT polypeptide as defined herein,having substantially the same biological activity as the amino acidsequences given in Table A1 of The Examples section. Preferably, thehybridising sequence is capable of hybridising to the complement of anyone of the nucleic acids given in Table A1 of The Examples section, orto a portion of any of these sequences, a portion being as definedabove, or the hybridising sequence is capable of hybridising to thecomplement of a nucleic acid encoding an orthologue or paralogue of anyone of the amino acid sequences given in Table A1 of The Examplessection. Even more preferably, the hybridising sequence is capable ofhybridising to the complement of a nucleic acid as represented by SEQ IDNO: 1 or to a portion thereof. Most preferably the hybridising sequenceis as represented by SEQ ID NO: 3.

Preferably, the hybridising sequence encodes a polypeptide with an aminoacid sequence which, when full-length and used in the construction of aphylogenetic tree, such as the one depicted in FIG. 2 clusters inincreasing order of preference with any of the polypeptides inphylogenetic class 1, class 2, class 3 and class 4 as set forth in TableB1.

Concerning MYB91 polypeptides, hybridising sequences useful in themethods of the invention encode an MYB91 polypeptide as defined herein,and have substantially the same biological activity as the polypeptidesequences given in Table A2 of Example 1. Preferably, the hybridisingsequence is capable of hybridising to any one of the nucleic acidsequences given in Table A2 of Example 1, or to a complement thereof, orto a portion of any of these sequences, a portion being as definedabove, or wherein the hybridising sequence is capable of hybridising toa nucleic acid sequence encoding an orthologue or paralogue of any oneof the polypeptide sequences given in Table A2 of Example 1, or to acomplement thereof. Preferably, the hybridising sequence is capable ofhybridising to a nucleic acid sequence encoding a polypeptide sequencecomprising (i) in increasing order of preference at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to a MYB DNA binding domain with an InterPro accession numberIPR014778, as represented by SEQ ID NO: 269; and (ii) in increasingorder of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or more amino acid sequence identity to a MYB DNAbinding domain with an InterPro accession number IPR014778, asrepresented by SEQ ID NO: 270; and (iii) in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to a Conserved Domain asrepresented by SEQ ID NO: 271. More preferably, the hybridising sequenceis capable of hybridising to a nucleic acid sequence encoding apolypeptide sequence having in increasing order of preference at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more aminoacid sequence identity to the MYB91 polypeptide as represented by SEQ IDNO: 221 or to any of the polypeptide sequences given in Table A2 herein.Most preferably, the hybridising sequence is capable of hybridising to anucleic acid sequence as represented by SEQ ID NO: 220 or to a portionthereof.

Concerning GASA polypeptides, hybridising sequences useful in themethods of the invention encode a GASA polypeptide as defined herein,having substantially the same biological activity as the amino acidsequences given in Table A3 of the Examples section. Preferably, thehybridising sequence is capable of hybridising to the complement of anyone of the nucleic acids given in Table A3 of the Examples section, orto a portion of any of these sequences, a portion being as definedabove, or the hybridising sequence is capable of hybridising to thecomplement of a nucleic acid encoding an orthologue or paralogue of anyone of the amino acid sequences given in Table A3 of the Examplessection. Most preferably, the hybridising sequence is capable ofhybridising to the complement of a nucleic acid as represented by SEQ IDNO: 275 or to a portion thereof.

Preferably, the hybridising sequence encodes a polypeptide with an aminoacid sequence which, when full-length and used in the construction of aphylogenetic tree, such as the one depicted in FIG. 9, clusters with thegroup of GASA polypeptides comprising the amino acid sequencerepresented by SEQ ID NO: 276 (or SEQ ID NO: 291 or SEQ ID NO: 292)rather than with any other group.

Concerning AUX/IAA polypeptides, hybridising sequences useful in themethods of the invention encode an AUX/IAA polypeptide as definedherein, having substantially the same biological activity as the aminoacid sequences given in Table A4 or in Table A5 of the Examples section.Preferably, the hybridising sequence is capable of hybridising to thecomplement of any one of the nucleic acids given in Table A4 or in TableA5 of the Examples section, or to a portion of any of these sequences, aportion being as defined above, or the hybridising sequence is capableof hybridising to the complement of a nucleic acid encoding anorthologue or paralogue of any one of the amino acid sequences given inTable A4 or in Table A5 of the Examples section. Most preferably, thehybridising sequence is capable of hybridising to the complement of anucleic acid as represented by SEQ ID NO: 431 or of SEQ ID NO: 737 or toa portion thereof.

Preferably, the hybridising sequence or its complementary sequenceencodes a polypeptide with an amino acid sequence comprising an AUX/IAAdomain (PFAM accession number PF2309, InterPro entry IPR003311).

In the case IAA14-like polypeptides, preferably, the hybridisingsequence encodes a polypeptide with an amino acid sequence which, whenfull-length and used in the construction of a phylogenetic tree, asdepicted in FIG. 1 in Remington et al. (Plant Physiol. 135, 1738-1752,2004), clusters with group A of the IAA14-like polypeptides, whichcomprises the amino acid sequence represented by SEQ ID NO: 738, ratherthan with any other group (see also FIG. 15).

Another nucleic acid variant useful in the methods of the invention is asplice variant encoding an ASPAT polypeptide, or an MYB91 polypeptide,or a GASA polypeptide, or an AUX/IAA polypeptide, as definedhereinabove, a splice variant being as defined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a splice variant of any one of the nucleic acidsequences given in Table A1 to A5 of The Examples section, or a splicevariant of a nucleic acid encoding an orthologue, paralogue or homologueof any of the amino acid sequences given in Table A1 to A5 of TheExamples section.

Concerning ASPAT polypeptides, preferred splice variants are splicevariants of a nucleic acid represented by SEQ ID NO: 1, or a splicevariant of a nucleic acid encoding an orthologue or paralogue of SEQ IDNO: 2. Preferably, the amino acid sequence encoded by the splicevariant, when used in the construction of a phylogenetic tree, such asthe one depicted in FIG. 2 clusters in increasing order of preferencewith any of the polypeptides in phylogenetic class 1, class 2, class 3and class 4 as set forth in Table B1.

Concerning MYB91 polypeptides, preferred splice variants are splicevariants of a nucleic acid sequence represented by SEQ ID NO: 220, or asplice variant of a nucleic acid sequence encoding an orthologue orparalogue of SEQ ID NO: 221. Preferably, the splice variant is a splicevariant of a nucleic acid sequence encoding a polypeptide sequencecomprising (i) in increasing order of preference at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to a MYB DNA binding domain with an InterPro accession numberIPR014778, as represented by SEQ ID NO: 269; and (ii) in increasingorder of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or more amino acid sequence identity to a MYB DNAbinding domain with an InterPro accession number IPR014778, asrepresented by SEQ ID NO: 270; and (iii) in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to a Conserved Domain asrepresented by SEQ ID NO: 271. More preferably, the splice variant is asplice variant of a nucleic acid sequence encoding a polypeptidesequence having in increasing order of preference at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acidsequence identity to the MYB91 polypeptide as represented by SEQ ID NO:221 or to any of the polypeptide sequences given in Table A2 herein.Most preferably, the splice variant is a splice variant of a nucleicacid sequence as represented by SEQ ID NO: 220, or of a nucleic acidsequence encoding a polypeptide sequence as represented by SEQ ID NO:221.

Concerning GASA polypeptides, preferred splice variants are splicevariants of a nucleic acid represented by SEQ ID NO: 275, or a splicevariant of a nucleic acid encoding an orthologue or paralogue of SEQ IDNO: 276. Preferably, the amino acid sequence encoded by the splicevariant, when used in the construction of a phylogenetic tree, such asthe one depicted in FIG. 93, clusters with the group of GASApolypeptides comprising the amino acid sequence represented by SEQ IDNO: 276 (or SEQ ID NO: 291 or SEQ ID NO: 292) rather than with any othergroup.

Concerning AUX/IAA polypeptides, preferred splice variants are splicevariants of a nucleic acid represented by SEQ ID NO: 431 or of SEQ IDNO: 737, or a splice variant of a nucleic acid encoding an orthologue orparalogue of SEQ ID NO: 432 or of SEQ ID NO: 738.

Preferably, the amino acid sequence encoded by the splice variantcomprises an AUX/IAA domain (PFAM accession number PF2309, InterProentry IPR003311).

In the case of IAA14-like polypeptides, preferably, the amino acidsequence encoded by the splice variant, when used in the construction ofa phylogenetic tree, as depicted in FIG. 1 in Remington et al. (PlantPhysiol. 135, 1738-1752, 2004), clusters with group A of the IAA14-likepolypeptides, which comprises the amino acid sequence represented by SEQID NO: 738, rather than with any other group (see also FIG. 15).

Another nucleic acid variant useful in performing the methods of theinvention is an allelic variant of a nucleic acid encoding an ASPATpolypeptide, or an MYB91 polypeptide, or a GASA polypeptide, or anAUX/IAA polypeptide, as defined hereinabove, an allelic variant being asdefined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant an allelic variant of any one of the nucleic acidsgiven in Table A1 to A5 of The Examples section, or comprisingintroducing and expressing in a plant an allelic variant of a nucleicacid encoding an orthologue, paralogue or homologue of any of the aminoacid sequences given in Table A1 to A5 of The Examples section.

Concerning ASPAT polypeptides, the polypeptides encoded by allelicvariants useful in the methods of the present invention havesubstantially the same biological activity as the ASPAT polypeptide ofSEQ ID NO: 2 and any of the amino acids depicted in Table A1 of TheExamples section. Allelic variants exist in nature, and encompassedwithin the methods of the present invention is the use of these naturalalleles. Preferably, the allelic variant is an allelic variant of SEQ IDNO: 1 or an allelic variant of a nucleic acid encoding an orthologue orparalogue of SEQ ID NO: 2. Preferably, the amino acid sequence encodedby the allelic variant, when used in the construction of a phylogenetictree, such as the one depicted in FIG. 2 clusters in increasing order ofpreference with any of the polypeptides in phylogenetic class 1, class2, class 3 and class 4 as set forth in Table B1.

Concerning MYB91 polypeptides, the allelic variants useful in themethods of the present invention have substantially the same biologicalactivity as the MYB91 polypeptide of SEQ ID NO: 221 and any of thepolypeptide sequences depicted in Table A2 of Example 1. Allelicvariants exist in nature, and encompassed within the methods of thepresent invention is the use of these natural alleles. Preferably, theallelic variant is an allelic variant of a polypeptide sequencecomprising (i) in increasing order of preference at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to a MYB DNA binding domain with an InterPro accession numberIPR014778, as represented by SEQ ID NO: 269; and (ii) in increasingorder of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or more amino acid sequence identity to a MYB DNAbinding domain with an InterPro accession number IPR014778, asrepresented by SEQ ID NO: 270; and (iii) in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to a Conserved Domain asrepresented by SEQ ID NO: 271. More preferably the allelic variant is anallelic variant encoding a polypeptide sequence having in increasingorder of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or more amino acid sequence identity to the MYB91polypeptide as represented by SEQ ID NO: 221 or to any of thepolypeptide sequences given in Table A2 herein. Most preferably, theallelic variant is an allelic variant of SEQ ID NO: 220 or an allelicvariant of a nucleic acid sequence encoding an orthologue or paralogueof SEQ ID NO: 221.

Concerning GASA polypeptides, the polypeptides encoded by allelicvariants useful in the methods of the present invention havesubstantially the same biological activity as the GASA polypeptide ofSEQ ID NO: 276 and any of the amino acids depicted in Table A3 of theExamples section. Allelic variants exist in nature, and encompassedwithin the methods of the present invention is the use of these naturalalleles. Preferably, the allelic variant is an allelic variant of SEQ IDNO: 275 or an allelic variant of a nucleic acid encoding an orthologueor paralogue of SEQ ID NO: 276. Preferably, the amino acid sequenceencoded by the allelic variant, when used in the construction of aphylogenetic tree, such as the one depicted in FIG. 9, clusters with thegroup of GASA polypeptides comprising the amino acid sequencerepresented by SEQ ID NO: 276 (or SEQ ID NO: 291 or SEQ ID NO: 292)rather than with any other group.

Concerning AUX/IAA polypeptides, the polypeptides encoded by allelicvariants useful in the methods of the present invention havesubstantially the same biological activity as the AUX/IAA polypeptide ofSEQ ID NO: 432 or of SEQ ID NO: 738 and any of the amino acids depictedin Table A4 or in Table A5 of the Examples section. Allelic variantsexist in nature, and encompassed within the methods of the presentinvention is the use of these natural alleles. Preferably, the allelicvariant is an allelic variant of SEQ ID NO: 431 or of SEQ ID NO: 737 oran allelic variant of a nucleic acid encoding an orthologue or paralogueof SEQ ID NO: 432 or of SEQ ID NO: 738. Preferably, the amino acidsequence encoded by the allelic variant comprises an AUX/IAA domain(PFAM accession number PF2309, InterPro entry IPR003311). In the case ofIAA14-like, preferably, the amino acid sequence encoded by the allelicvariant, when used in the construction of a phylogenetic tree, asdepicted in FIG. 1 in Remington et al. (Plant Physiol. 135, 1738-1752,2004), clusters with group A of the IAA14-like polypeptides, whichcomprises the amino acid sequence represented by SEQ ID NO: 738, ratherthan with any other group (see also FIG. 15).

Gene shuffling or directed evolution may also be used to generatevariants of nucleic acids encoding ASPAT polypeptides, or MYB91polypeptides, GASA polypeptides, AUX/IAA polypeptides, or as definedabove; the term “gene shuffling” being as defined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a variant of any one of the nucleic acid sequencesgiven in Table A1 to A5 of The Examples section, or comprisingintroducing and expressing in a plant a variant of a nucleic acidencoding an orthologue, paralogue or homologue of any of the amino acidsequences given in Table A1 to A5 of The Examples section, which variantnucleic acid is obtained by gene shuffling.

Concerning ASPAT polypeptides, preferably, the amino acid sequenceencoded by the variant nucleic acid obtained by gene shuffling, whenused in the construction of a phylogenetic tree such as the one depictedin FIG. 2 clusters in increasing order of preference with any of thepolypeptides in phylogenetic class 1, class 2, class 3 and class 4 asset forth in Table B1.

Concerning MYB91 polypeptides, preferably, the variant nucleic acidsequence obtained by gene shuffling encodes a polypeptide sequencecomprising (i) in increasing order of preference at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to a MYB DNA binding domain with an InterPro accession numberIPR014778, as represented by SEQ ID NO: 269; and (ii) in increasingorder of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%_(,) 99% or more amino acid sequence identity to a MYB DNAbinding domain with an InterPro accession number IPR014778, asrepresented by SEQ ID NO: 270; and (iii) in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to a Conserved Domain asrepresented by SEQ ID NO: 271. More preferably, the variant nucleic acidsequence obtained by gene shuffling encodes a polypeptide sequencehaving in increasing order of preference at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to the MYB91 polypeptide as represented by SEQ ID NO: 221 or toany of the polypeptide sequences given in Table A1 herein. Mostpreferably, the nucleic acid sequence obtained by gene shuffling encodesa polypeptide sequence as represented by SEQ ID NO: 221.

Concerning GASA polypeptides, preferably, the amino acid sequenceencoded by the variant nucleic acid obtained by gene shuffling, whenused in the construction of a phylogenetic tree such as the one depictedin FIG. 9, clusters with the group of GASA polypeptides comprising theamino acid sequence represented by SEQ ID NO: 276 (or SEQ ID NO: 291 orSEQ ID NO: 292) rather than with any other group.

In the case of IAA14-like polypeptides, preferably, the amino acidsequence encoded by the variant nucleic acid obtained by gene shuffling,when used in the construction of a phylogenetic tree, as depicted inFIG. 1 in Remington et al. (Plant Physiol. 135, 1738-1752, 2004),clusters with group A of the IAA14-like polypeptides, which comprisesthe amino acid sequence represented by SEQ ID NO: 738, rather than withany other group (see also FIG. 15).

Furthermore, nucleic acid variants may also be obtained by site-directedmutagenesis. Several methods are available to achieve site-directedmutagenesis, the most common being PCR based methods (Current Protocolsin Molecular Biology. Wiley Eds.).

Nucleic acids encoding ASPAT polypeptides may be derived from anynatural or artificial source. The nucleic acid may be modified from itsnative form in composition and/or genomic environment through deliberatehuman manipulation. Preferably the ASPAT polypeptide-encoding nucleicacid is from a plant, further preferably from a monocotyledonous plant,more preferably from the family Poaceae, most preferably the nucleicacid is from Oryza sativa.

Advantageously, the invention also provides hitherto unknownASPAT-encoding nucleic acids and ASPAT polypeptides.

According to a further embodiment of the present invention, there istherefore provided an isolated nucleic acid molecule selected from:

-   -   (i) a nucleic acid represented by any one of SEQ ID NO: 81, 147,        153, 183 and 185;    -   (ii) the complement of a nucleic acid represented by any one of        SEQ ID NO: 81, 147, 153, 183 and 185;    -   (iii) a nucleic acid encoding the polypeptide as represented by        any one of SEQ ID NO: 82, 148, 154, 184 and 186, preferably as a        result of the degeneracy of the genetic code, said isolated        nucleic acid can be derived from a polypeptide sequence as        represented by any one of SEQ ID NO: 82, 148, 154, 184 and 186        and further preferably confers enhanced yield-related traits        relative to control plants;    -   (iv) a nucleic acid having, in increasing order of preference at        least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,        41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any        of the nucleic acid sequences of Table A1 and further preferably        conferring enhanced yield-related traits relative to control        plants;    -   (v) a nucleic acid molecule which hybridizes with a nucleic acid        molecule of (i) to    -   (iv) under stringent hybridization conditions and preferably        confers enhanced yield-related traits relative to control        plants;    -   (vi) a nucleic acid encoding an ASPAT polypeptide having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        amino acid sequence represented by any one of SEQ ID NO: 82,        148, 154, 184 and 186 and any of the other amino acid sequences        in Table A1 and preferably conferring enhanced yield-related        traits relative to control plants.

According to a further embodiment of the present invention, there isalso provided an isolated polypeptide selected from:

-   -   (i) an amino acid sequence represented by any one of SEQ ID NO:        82, 148, 154, 184 and 186;    -   (ii) an amino acid sequence having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the amino acid sequence        represented by any one of SEQ ID NO: 82, 148, 154, 184 and 186,        and any of the other amino acid sequences in Table A1 and        preferably conferring enhanced yield-related traits relative to        control plants. (iii) derivatives of any of the amino acid        sequences given in (i) or (ii) above.

Nucleic acid sequences encoding MYB91 polypeptides may be derived fromany natural or artificial source. The nucleic acid sequence may bemodified from its native form in composition and/or genomic environmentthrough deliberate human manipulation. The nucleic acid sequenceencoding an MYB91 polypeptide is from a plant, further preferably from adicotyledonous plant, more preferably from the family Salicaceae, mostpreferably the nucleic acid sequence is from Populus trichocarpa.

Nucleic acids encoding GASA polypeptides may be derived from any naturalor artificial source. The nucleic acid may be modified from its nativeform in composition and/or genomic environment through deliberate humanmanipulation. Preferably the GASA polypeptide-encoding nucleic acid isfrom a plant, further preferably from a dicotyledonous plant, morepreferably from the family Solanaceae, most preferably the nucleic acidis from Solanum lycopersicum. Alternatively, the GASApolypeptide-encoding nucleic acid is from the family Salicaceae,preferably from Populus sp.

Nucleic acids encoding AUX/IAA polypeptides may be derived from anynatural or artificial source. The nucleic acid may be modified from itsnative form in composition and/or genomic environment through deliberatehuman manipulation. Preferably the IAA14-like polypeptide-encodingnucleic acid is from a plant, further preferably from a monocotyledonousor a dicotyledonous plan, more preferably from the family Poaceae orBrassicaceae, most preferably the nucleic acid is from Oryza sativa orfrom Arabidopsis thaliana.

Performance of the methods of the invention gives plants having enhancedyield-related traits. In particular performance of the methods of theinvention gives plants having increased yield, especially increased seedyield relative to control plants. The terms “yield” and “seed yield” aredescribed in more detail in the “definitions” section herein.

Reference herein to enhanced yield-related traits is taken to mean anincrease in biomass (weight) of one or more parts of a plant, which mayinclude aboveground (harvestable) parts and/or (harvestable) parts belowground. In particular, such harvestable parts are seeds, and performanceof the methods of the invention results in plants having increased seedyield relative to the seed yield of control plants. Concerning GASApolypeptides, It should be noted that the plants with modulatedexpression of a nucleic acid encoding a GASA polypeptide according tothe methods of this invention did not show significant changes inbranching properties compared to the control plants.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants established persquare meter, an increase in the number of ears per plant, an increasein the number of rows, number of kernels per row, kernel weight,thousand kernel weight, ear length/diameter, increase in the seedfilling rate (which is the number of filled seeds divided by the totalnumber of seeds and multiplied by 100), among others. Taking rice as anexample, a yield increase may manifest itself as an increase in one ormore of the following: number of plants per square meter, number ofpanicles per plant, number of spikelets per panicle, number of flowers(florets) per panicle (which is expressed as a ratio of the number offilled seeds over the number of primary panicles), increase in the seedfilling rate (which is the number of filled seeds divided by the totalnumber of seeds and multiplied by 100), increase in thousand kernelweight, among others.

The present invention provides a method for increasing yield, especiallyseed yield of plants, relative to control plants, which method comprisesmodulating expression in a plant of a nucleic acid encoding an ASPATpolypeptide, or a GASA polypeptide, or an AUX/IAA polypeptide, asdefined herein.

The present invention also provides a method for increasingyield-related traits of plants relative to control plants, which methodcomprises increasing expression in a plant of a nucleic acid sequenceencoding an MYB91 polypeptide as defined herein.

Since the transgenic plants according to the present invention haveincreased yield and/or increased yield-related traits, it is likely thatthese plants exhibit an increased growth rate (during at least part oftheir life cycle), relative to the growth rate of control plants at acorresponding stage in their life cycle.

The increased growth rate may be specific to one or more parts of aplant (including seeds), or may be throughout substantially the wholeplant. Plants having an increased growth rate may have a shorter lifecycle. The life cycle of a plant may be taken to mean the time needed togrow from a dry mature seed up to the stage where the plant has produceddry mature seeds, similar to the starting material. This life cycle maybe influenced by factors such as early vigour, growth rate, greennessindex, flowering time and speed of seed maturation. The increase ingrowth rate may take place at one or more stages in the life cycle of aplant or during substantially the whole plant life cycle. Increasedgrowth rate during the early stages in the life cycle of a plant mayreflect increased (early) vigour. The increase in growth rate may alterthe harvest cycle of a plant allowing plants to be sown later and/orharvested sooner than would otherwise be possible (a similar effect maybe obtained with earlier flowering time; delayed flowering is usuallynot a desirede trait in crops). If the growth rate is sufficientlyincreased, it may allow for the further sowing of seeds of the sameplant species (for example sowing and harvesting of rice plants followedby sowing and harvesting of further rice plants all within oneconventional growing period). Similarly, if the growth rate issufficiently increased, it may allow for the further sowing of seeds ofdifferent plants species (for example the sowing and harvesting of cornplants followed by, for example, the sowing and optional harvesting ofsoybean, potato or any other suitable plant). Harvesting additionaltimes from the same rootstock in the case of some crop plants may alsobe possible. Altering the harvest cycle of a plant may lead to anincrease in annual biomass production per acre (due to an increase inthe number of times (say in a year) that any particular plant may begrown and harvested). An increase in growth rate may also allow for thecultivation of transgenic plants in a wider geographical area than theirwild-type counterparts, since the territorial limitations for growing acrop are often determined by adverse environmental conditions either atthe time of planting (early season) or at the time of harvesting (lateseason). Such adverse conditions may be avoided if the harvest cycle isshortened. The growth rate may be determined by deriving variousparameters from growth curves, such parameters may be: T-Mid (the timetaken for plants to reach 50% of their maximal size) and T-90 (timetaken for plants to reach 90% of their maximal size), amongst others.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having an increased growthrate relative to control plants. Therefore, according to the presentinvention, there is provided a method for increasing the growth rate ofplants, which method comprises modulating expression in a plant of anucleic acid encoding an ASPAT polypeptide, or an MYB91 polypeptide, ora GASA polypeptide, or an AUX/IAA polypeptide, as defined herein.

Increased yield-related traits occur whether the plant is undernon-stress conditions or whether the plant is exposed to variousstresses compared to control plants grown under comparable conditions.Plants typically respond to exposure to stress by growing more slowly.In conditions of severe stress, the plant may even stop growingaltogether. Mild stress on the other hand is defined herein as being anystress to which a plant is exposed which does not result in the plantceasing to grow altogether without the capacity to resume growth. Mildstress in the sense of the invention leads to a reduction in the growthof the stressed plants of less than 40%, 35% or 30%, preferably lessthan 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or10% or less in comparison to the control plant under non-stressconditions. Due to advances in agricultural practices (irrigation,fertilization, and/or pesticide treatments) severe stresses are notoften encountered in cultivated crop plants. As a consequence, thecompromised growth induced by mild stress is often an undesirablefeature for agriculture. Mild stresses are the everyday biotic and/orabiotic (environmental) stresses to which a plant is exposed. Abioticstresses may be due to drought or excess water, anaerobic stress, saltstress, chemical toxicity, oxidative stress and hot, cold or freezingtemperatures. The abiotic stress may be an osmotic stress caused by awater stress (particularly due to drought), salt stress, oxidativestress or an ionic stress. Biotic stresses are typically those stressescaused by pathogens, such as bacteria, viruses, fungi, nematodes, andinsects. The term “non-stress” conditions as used herein are thoseenvironmental conditions that allow optimal growth of plants. Personsskilled in the art are aware of normal soil conditions and climaticconditions for a given location.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild stress conditions having increasedyield-related traits, relative to control plants grown under comparableconditions. Therefore, according to the present invention, there isprovided a method for increasing yield-related traits in plants grownunder non-stress conditions or under mild stress conditions, whichmethod comprises increasing expression in a plant of a nucleic acidsequence encoding an MYB91 polypeptide.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signalling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location. Plants withoptimal growth conditions, (grown under non-stress conditions) typicallyyield in increasing order of preference at least 97%, 95%, 92%, 90%,87%, 85%, 83%, 80%, 77% or 75% of the average production of such plantin a given environment. Average production may be calculated on harvestand/or season basis. Persons skilled in the art are aware of averageyield productions of a crop.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under non-stress conditions or undermild drought conditions, which method comprises modulating expression ina plant of a nucleic acid encoding an ASPAT polypeptide, or an MYB91polypeptide, or a GASA polypeptide, or an AUX/IAA polypeptide.

The term “abiotic stress” as defined herein is taken to mean any one ormore of: water stress (due to drought or excess water), anaerobicstress, salt stress, temperature stress (due to hot, cold or freezingtemperatures), chemical toxicity stress and oxidative stress. Accordingto one aspect of the invention, the abiotic stress is an osmotic stress,selected from water stress, salt stress, oxidative stress and ionicstress. Preferably, the water stress is drought stress. The term saltstress is not restricted to common salt (NaCl), but may be any stresscaused by one or more of: NaCl, KCl, LiCl, MgCl₂, CaCl₂, amongst others.

Performance of the methods of the invention gives plants grown underconditions of salt stress, increased yield relative to control plantsgrown under comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of salt stress, which method comprises modulatingexpression in a plant of a nucleic acid encoding an ASPAT polypeptide,or a GASA polypeptide, or an AUX/IAA polypeptide. The term salt stressis not restricted to common salt (NaCl), but may be any one or more of:NaCl, KCl, LiCl, MgCl₂, CaCl₂, amongst others.

Another example of abiotic environmental stress is the reducedavailability of one or more nutrients that need to be assimilated by theplants for growth and development. Because of the strong influence ofnutrition utilization efficiency on plant yield and product quality, ahuge amount of fertilizer is poured onto fields to optimize plant growthand quality. Productivity of plants ordinarily is limited by threeprimary nutrients, phosphorous, potassium and nitrogen, which is usuallythe rate-limiting element in plant growth of these three. Therefore themajor nutritional element required for plant growth is nitrogen (N). Itis a constituent of numerous important compounds found in living cells,including amino acids, proteins (enzymes), nucleic acids, andchlorophyll. 1.5% to 2% of plant dry matter is nitrogen andapproximately 16% of total plant protein. Thus, nitrogen availability isa major limiting factor for crop plant growth and production (Frink etal. (1999) Proc Natl Acad Sci USA 96(4): 1175-1180), and has as well amajor impact on protein accumulation and amino acid composition.Therefore, of great interest are crop plants with increasedyield-related traits, when grown under nitrogen-limiting conditions.

Performance of the methods of the invention gives plants grown underconditions of nutrient deficiency, particularly under conditions ofnitrogen deficiency, increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of nutrient deficiency, which method comprisesmodulating expression in a plant of a nucleic acid encoding an ASPATpolypeptide, or a GASA polypeptide, or an AUX/IAA polypeptide. Nutrientdeficiency may result from a lack of nutrients such as nitrogen,phosphates and other phosphorous-containing compounds, potassium,calcium, cadmium, magnesium, manganese, iron and boron, amongst others.

Performance of the methods of the invention gives plants grown underconditions of reduced nutrient availability, particularly underconditions of reduced nitrogen availability, having increasedyield-related traits relative to control plants grown under comparableconditions. Therefore, according to the present invention, there isprovided a method for increasing yield-related traits in plants grownunder conditions of reduced nutrient availablity, preferably reducednitrogen availability, which method comprises increasing expression in aplant of a nucleic acid sequence encoding an MYB91 polypeptide. Reducednutrient availability may result from a deficiency or excess ofnutrients such as nitrogen, phosphates and other phosphorous-containingcompounds, potassium, calcium, cadmium, magnesium, manganese, iron andboron, amongst others. Preferably, reduced nutrient availablity isreduced nitrogen availability.

Performance of the methods of the invention gives plants havingincreased yield-related traits, under abiotic stress conditions relativeto control plants grown in comparable stress conditions. Therefore,according to the present invention, there is provided a method forincreasing yield-related traits, in plants grown under abiotic stressconditions, which method comprises increasing expression in a plant of anucleic acid sequence encoding an MYB91 polypeptide. According to oneaspect of the invention, the abiotic stress is an osmotic stress,selected from one or more of the following: water stress, salt stress,oxidative stress and ionic stress.

The present invention encompasses plants or parts thereof (includingseeds) or cells thereof obtainable by the methods according to thepresent invention. The plants or parts thereof comprise a nucleic acidtransgene encoding an ASPAT polypeptide, or an MYB91 polypeptide, or aGASA polypeptide, or an AUX/IAA polypeptide, as defined above, operablylinked to a promoter functioning in plants.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression in plants of nucleic acids encoding ASPATpolypeptides, or MYB91 polypeptides, or GASA polypeptides, or AUX/IAApolypeptides, as defined herein. The gene constructs may be insertedinto vectors, which may be commercially available, suitable fortransforming into plants and suitable for expression of the gene ofinterest in the transformed cells. The invention also provides use of agene construct as defined herein in the methods of the invention.

More specifically, the present invention provides a constructcomprising:

-   -   (a) a nucleic acid encoding an ASPAT polypeptide, or an MYB91        polypeptide, or a GASA polypeptide, or an AUX/IAA polypeptide,        as defined above;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.

Preferably, the nucleic acid encoding is an ASPAT polypeptide, or anMYB91 polypeptide, or a GASA polypeptide, or an AUX/IAA polypeptide, asdefined above. The term “control sequence” and “termination sequence”are as defined herein.

Concerning MYB91 polypeptides, preferably, one of the control sequencesof a construct is a constitutive promoter isolated from a plant genome.An example of a constitutive promoter is a GOS2 promoter, preferably aGOS2 promoter from rice, most preferably a GOS2 sequence as representedby SEQ ID NO: 272.

Plants are transformed with a vector comprising any of the nucleic acidsdescribed above. The skilled artisan is well aware of the geneticelements that must be present on the vector in order to successfullytransform, select and propagate host cells containing the sequence ofinterest. The sequence of interest is operably linked to one or morecontrol sequences (at least to a promoter).

Advantageously, any type of promoter, whether natural or synthetic, maybe used to drive expression of the nucleic acid sequence, but preferablythe promoter is of plant origin. A constitutive promoter is particularlyuseful in the methods. Preferably the constitutive promoter is also aubiquitous promoter of medium strength. See the “Definitions” sectionherein for definitions of the various promoter types. Concerning ASPATpolypeptides, also useful in the methods of the invention is a greentissue-specific promoter.

Concerning MYB91 polypeptides, advantageously, any type of promoter,whether natural or synthetic, may be used to increase expression of thenucleic acid sequence. A constitutive promoter is particularly useful inthe methods, preferably a constitutive promoter isolated from a plantgenome. The plant constitutive promoter drives expression of a codingsequence at a level that is in all instances below that obtained underthe control of a 35S CaMV viral promoter. An example of such a promoteris a GOS2 promoter as represented by SEQ ID NO: 272.

Concerning MYB91 polypeptides, organ-specific promoters, for example forpreferred expression in leaves, stems, tubers, meristems, seeds, areuseful in performing the methods of the invention.Developmentally-regulated and inducible promoters are also useful inperforming the methods of the invention. See the “Definitions” sectionherein for definitions of the various promoter types.

Concerning ASPAT polypeptides, it should be clear that the applicabilityof the present invention is not restricted to the ASPATpolypeptide-encoding nucleic acid represented by SEQ ID NO: 1, nor isthe applicability of the invention restricted to expression of an ASPATpolypeptide-encoding nucleic acid when driven by a constitutivepromoter, or when driven by a green tissue-specific promoter.

The constitutive promoter is preferably a medium strength promoter, morepreferably selected from a plant derived promoter, such as a GOS2promoter, more preferably is the promoter GOS2 promoter from rice.Further preferably the constitutive promoter is represented by a nucleicacid sequence substantially similar to SEQ ID NO: 218, most preferablythe constitutive promoter is as represented by SEQ ID NO: 218. See the“Definitions” section herein for further examples of constitutivepromoters.

According to another preferred feature of the invention, the nucleicacid encoding an ASPAT polypeptide is operably linked to a greentissue-specific promoter. The green tissue-specific promoter ispreferably a promoter of the a Protochlorophyllide reductase (PR) gene,more preferably the PR promoter is from rice, further preferably the PRpromoter is represented by a nucleic acid sequence substantially similarto SEQ ID NO: 219, most preferably the promoter is as represented by SEQID NO: 219. Examples of other green tissue-specific promoters which mayalso be used to perform the methods of the invention are shown in Table3 in the “Definitions” section above.

Concerning MYB91 polypeptides, it should be clear that the applicabilityof the present invention is not restricted to a nucleic acid sequenceencoding the MYB91 polypeptide, as represented by SEQ ID NO: 220, nor isthe applicability of the invention restricted to expression of an MYB91polypeptide-encoding nucleic acid sequence when driven by a constitituvepromoter.

Concerning GASA polypeptides, it should be clear that the applicabilityof the present invention is not restricted to the GASApolypeptide-encoding nucleic acid represented by SEQ ID NO: 275 or SEQID NO: 361, nor is the applicability of the invention restricted toexpression of a GASA polypeptide-encoding nucleic acid when driven by aconstitutive promoter.

The constitutive promoter is preferably a medium strength promoter, morepreferably selected from a plant derived promoter, such as a GOS2promoter, more preferably is the promoter a GOS2 promoter from rice.Further preferably the constitutive promoter is represented by a nucleicacid sequence substantially similar to SEQ ID NO: 290, most preferablythe constitutive promoter is as represented by SEQ ID NO: 290. See the“Definitions” section herein for further examples of constitutivepromoters.

Optionally, one or more terminator sequences may be used in theconstruct introduced into a plant. Preferably, the construct comprisesan expression cassette comprising a GOS2 promoter and the nucleic acidencoding the GASA polypeptide.

Concerning AUX/IAA polypeptides, it should be clear that theapplicability of the present invention is not restricted to the AUX/IAApolypeptide-encoding nucleic acid represented by SEQ ID NO: 431 or bySEQ ID NO: 737, nor is the applicability of the invention restricted toexpression of an AUX/IAA polypeptide-encoding nucleic acid when drivenby a constitutive promoter.

The constitutive promoter is preferably a medium strength promoter, morepreferably selected from a plant derived promoter, such as a GOS2promoter, more preferably is the promoter GOS2 promoter from rice.Further preferably the constitutive promoter is represented by a nucleicacid sequence substantially similar to SEQ ID NO: 669, most preferablythe constitutive promoter is as represented by SEQ ID NO: 669. See the“Definitions” section herein for further examples of constitutivepromoters.

Alternatively, the constitutive promoter is preferably a weakconstitutive promoter, more preferably selected from a plant derivedpromoter, such as a High Mobility Group Protein (HMGP) promoter, morepreferably is the promoter HMGP promoter from rice. Further preferablythe constitutive promoter is represented by a nucleic acid sequencesubstantially similar to SEQ ID NO: 747, most preferably theconstitutive promoter is as represented by SEQ ID NO: 747. See the“Definitions” section herein for further examples of constitutivepromoters.

Optionally, one or more terminator sequences may be used in theconstruct introduced into a plant. Preferably, the construct comprisesan expression cassette comprising a GOS2 or a HMGP promoter,substantially similar to SEQ ID NO: 669 or to SEQ ID NO: 747respectively, and the nucleic acid encoding the AUX/IAA polypeptide.

Additional regulatory elements may include transcriptional as well astranslational enhancers. Those skilled in the art will be aware ofterminator and enhancer sequences that may be suitable for use inperforming the invention. An intron sequence may also be added to the 5′untranslated region (UTR) or in the coding sequence to increase theamount of the mature message that accumulates in the cytosol, asdescribed in the definitions section. Other control sequences (besidespromoter, enhancer, silencer, intron sequences, 3′UTR and/or 5′UTRregions) may be protein and/or RNA stabilizing elements. Such sequenceswould be known or may readily be obtained by a person skilled in theart.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1.

For the detection of the successful transfer of the nucleic acidsequences as used in the methods of the invention and/or selection oftransgenic plants comprising these nucleic acids, it is advantageous touse marker genes (or reporter genes). Therefore, the genetic constructmay optionally comprise a selectable marker gene. Selectable markers aredescribed in more detail in the “definitions” section herein. The markergenes may be removed or excised from the transgenic cell once they areno longer needed. Techniques for marker removal are known in the art,useful techniques are described above in the definitions section.

It is known that upon stable or transient integration of nucleic acidsequences into plant cells, only a minority of the cells takes up theforeign DNA and, if desired, integrates it into its genome, depending onthe expression vector used and the transfection technique used. Toidentify and select these integrants, a gene coding for a selectablemarker (such as the ones described above) is usually introduced into thehost cells together with the gene of interest. These markers can forexample be used in mutants in which these genes are not functional by,for example, deletion by conventional methods. Furthermore, nucleic acidsequence molecules encoding a selectable marker can be introduced into ahost cell on the same vector that comprises the sequence encoding thepolypeptides of the invention or used in the methods of the invention,or else in a separate vector. Cells which have been stably transfectedwith the introduced nucleic acid sequence can be identified for exampleby selection (for example, cells which have integrated the selectablemarker survive whereas the other cells die). The marker genes may beremoved or excised from the transgenic cell once they are no longerneeded. Techniques for marker gene removal are known in the art, usefultechniques are described above in the definitions section.

The invention also provides a method for the production of transgenicplants having enhanced yield-related traits relative to control plants,comprising introduction and expression in a plant of any nucleic acidencoding an ASPAT polypeptide, or an MYB91 polypeptide, or a GASApolypeptide, or an AUX/IAA polypeptide, as defined hereinabove.

More specifically, the present invention provides a method for theproduction of transgenic plants having enhanced yield-related traits,particularly increased seed yield, which method comprises:

-   -   (i) introducing and expressing in a plant, plant part, or plant        cell a nucleic acid encoding an ASPAT polypeptide, or an MYB91        polypeptide, or a GASA polypeptide, or an AUX/IAA polypeptide;        and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding an ASPAT polypeptide, or an MYB91 polypeptide, or a GASApolypeptide, or an AUX/IAA polypeptide, as defined herein.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation. The term “transformation” is described in more detail inthe “definitions” section herein.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation. The term “transformation” is described in more detail inthe “definitions” section herein.

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the above-mentioned publications by S. D. Kung and R. Wu, Potrykus orHofgen and Willmitzer.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques. The generated transformed organisms may take a variety offorms. For example, they may be chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g., all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissues (e.g., in plants, a transformed rootstock graftedto an untransformed scion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding an ASPAT polypeptide, or an MYB91 polypeptide, or a GASApolypeptide, or an AUX/IAA polypeptide, as defined hereinabove.Preferred host cells according to the invention are plant cells. Hostplants for the nucleic acids or the vector used in the method accordingto the invention, the expression cassette or construct or vector are, inprinciple, advantageously all plants, which are capable of synthesizingthe polypeptides used in the inventive method.

The methods of the invention are advantageously applicable to any plant.Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, linseed, cotton, tomato, potato and tobacco.Further preferably, the plant is a monocotyledonous plant. Examples ofmonocotyledonous plants include sugarcane. More preferably the plant isa cereal. Examples of cereals include rice, maize, wheat, barley,millet, rye, triticale, sorghum, emmer, spelt, secale, einkorn, teff,milo and oats.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes,tubers and bulbs, which harvestable parts comprise a recombinant nucleicacid encoding an ASPAT polypeptide, or an MYB91 polypeptide, or a GASApolypeptide, or an AUX/IAA polypeptide. The invention furthermorerelates to products derived, preferably directly derived, from aharvestable part of such a plant, such as dry pellets or powders, oil,fat and fatty acids, starch or proteins.

According to a preferred feature of the invention, the modulatedexpression is increased expression. Methods for increasing expression ofnucleic acids or genes, or gene products, are well documented in the artand examples are provided in the definitions section.

As mentioned above, a preferred method for modulating expression of anucleic acid encoding an ASPAT polypeptide, or an MYB91 polypeptide, ora GASA polypeptide, or an AUX/IAA polypeptide, is by introducing andexpressing in a plant a nucleic acid encoding an ASPAT polypeptide, oran MYB91 polypeptide, or a GASA polypeptide, or an AUX/IAA polypeptide;however the effects of performing the method, i.e. enhancingyield-related traits may also be achieved using other well knowntechniques, including but not limited to T-DNA activation tagging,TILLING, homologous recombination. A description of these techniques isprovided in the definitions section.

The present invention also encompasses use of nucleic acids encodingASPAT polypeptides, or GASA polypeptides, or AUX/IAA polypeptides, asdescribed herein and use of these ASPAT polypeptides, or GASApolypeptides, or AUX/IAA polypeptides, in enhancing any of theaforementioned yield-related traits in plants.

The present invention also encompasses use of nucleic acid sequencesencoding MYB91 polypeptides as described herein and use of these MYB91polypeptides in increasing any of the aforementioned yield-relatedtraits in plants, under normal growth conditions, under abiotic stressgrowth (preferably osmotic stress growth conditions) conditions, andunder growth conditions of reduced nutrient availability, preferablyunder conditions of reduced nitrogen availability.

Nucleic acids encoding an ASPAT polypeptide, or an MYB91 polypeptide, ora GASA polypeptide, or an AUX/IAA polypeptide, described herein, or theASPAT polypeptides, or MYB91 polypeptides, or GASA polypeptides, orAUX/IAA polypeptides, themselves, may find use in breeding programmes inwhich a DNA marker is identified which may be genetically linked to agene encoding an ASPAT polypeptide, or an MYB91 polypeptide, or a GASApolypeptide, or an AUX/IAA polypeptide. The nucleic acids/genes, or theASPAT polypeptides themselves may be used to define a molecular marker.This DNA or protein marker may then be used in breeding programmes toselect plants having enhanced yield-related traits as definedhereinabove in the methods of the invention.

Allelic variants of a nucleic acid/gene encoding an ASPAT polypeptide,or an MYB91 polypeptide, or a GASA polypeptide, or an AUX/IAApolypeptide may also find use in marker-assisted breeding programmes.Such breeding programmes sometimes require introduction of allelicvariation by mutagenic treatment of the plants, using for example EMSmutagenesis; alternatively, the programme may start with a collection ofallelic variants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place, for example, byPCR. This is followed by a step for selection of superior allelicvariants of the sequence in question and which give increased yieldand/or yield-related traits. Selection is typically carried out bymonitoring growth performance of plants containing different allelicvariants of the sequence in question. Growth performance may bemonitored in a greenhouse or in the field. Further optional stepsinclude crossing plants in which the superior allelic variant wasidentified with another plant. This could be used, for example, to makea combination of interesting phenotypic features.

Nucleic acids encoding ASPAT polypeptides, or MYB91 polypeptides, orGASA polypeptides, or AUX/IAA polypeptides, may also be used as probesfor genetically and physically mapping the genes that they are a partof, and as markers for traits linked to those genes. Such informationmay be useful in plant breeding in order to develop lines with desiredphenotypes. Such use of nucleic acids encoding an ASPAT polypeptide, oran MYB91 polypeptide, or a GASA polypeptide, or an AUX/IAA polypeptide,requires only a nucleic acid sequence of at least 15 nucleotides inlength. The encoding nucleic acids may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Sambrook J, FritschEF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) ofrestriction-digested plant genomic DNA may be probed with the encodingnucleic acids encoding an ASPAT polypeptide, or an MYB91 polypeptide, ora GASA polypeptide, or an AUX/IAA polypeptide. The resulting bandingpatterns may then be subjected to genetic analyses using computerprograms such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) inorder to construct a genetic map. In addition, the nucleic acids may beused to probe Southern blots containing restriction endonuclease-treatedgenomic DNAs of a set of individuals representing parent and progeny ofa defined genetic cross. Segregation of the DNA polymorphisms is notedand used to calculate the position of the nucleic acid encoding an ASPATpolypeptide, or an MYB91 polypeptide, or a GASA polypeptide, or anAUX/IAA polypeptide, in the genetic map previously obtained using thispopulation (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridisation (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favour useof large clones (several kb to several hundred kb; see Laan et al.(1995) Genome Res. 5:13-20), improvements in sensitivity may allowperformance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin.Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffieldet al. (1993) Genomics 16:325-332), allele-specific ligation (Landegrenet al. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

Concerning ASPAT polypeptides, concerning GASA polypeptides, or anAUX/IAA polypeptide, the methods according to the present inventionresult in plants having enhanced yield-related traits, as describedhereinbefore. These traits may also be combined with other economicallyadvantageous traits, such as further yield-enhancing traits, toleranceto other abiotic and biotic stresses, traits modifying variousarchitectural features and/or biochemical and/or physiological features.

Concerning MYB91 polypeptides, the methods according to the presentinvention result in plants having increased yield-related traits, asdescribed hereinbefore. These traits may also be combined with othereconomically advantageous traits, such as further yield-increasingtraits, tolerance to abiotic and biotic stresses, tolerance toherbicides, insectides, traits modifying various architectural featuresand/or biochemical and/or physiological features.

Items 1. Aspartate AminoTransferase (ASPAT)

-   1. A method for enhancing yield-related traits in plants relative to    control plants, comprising modulating expression in a plant of a    nucleic acid encoding an ASPAT (Aspartate Aminotransferase)    polypeptide comprising an Aminotransferase class I and II    (Aminotran_(—)1_(—)2) domain (Interpro accession number: IPR004839;    pfam accession number: PF00155), and optionally selecting plants    having enhanced yield-related traits-   2. Method according to item 1, wherein said ASPAT polypeptide    comprising one or more of the following motifs having at least 50%,    51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,    64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,    77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,    90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any one    or more of the following motif:

(i) Motif 1: NPTG, (SEQ ID NO: 207) (ii) Motif 2: IVLLHACAHNPTGVDPT,(SEQ ID NO: 208) (iii) Motif 3: SRLLILCSPSNPTGSVY (SEQ ID NO: 209)wherein any amino acid residue maybe substituted by a conserved aminoacid.

-   3. Method according to item 1 or 2, wherein said modulated    expression is effected by introducing and expressing in a plant a    nucleic acid encoding an ASPAT polypeptide.-   4. Method according to any one of items 1 to 3, wherein said nucleic    acid encoding an ASPAT polypeptide encodes any one of the proteins    listed in Table A or is a portion of such a nucleic acid, or a    nucleic acid capable of hybridising with such a nucleic acid.-   5. Method according to any one of items 1 to 4, wherein said nucleic    acid sequence encodes an orthologue or paralogue of any of the    proteins given in Table A1.-   6. Method according to any preceding item, wherein said enhanced    yield-related traits comprise increased yield, preferably increased    biomass and/or increased seed yield relative to control plants.-   7. Method according to any one of items 1 to 6, wherein said    enhanced yield-related traits are obtained under non-stress    conditions.-   8. Method according to any one of items 1 to 6, wherein said    enhanced yield-related traits are obtained under conditions of    drought stress, salt stress or nitrogen deficiency.-   9. Method according to any one of items 3 to 8, wherein said nucleic    acid is operably linked to a constitutive promoter, preferably to a    GOS2 promoter, most preferably to a GOS2 promoter from rice.-   10. Method according to any one of items 3 to 8, wherein said    nucleic acid is operably linked to a green tissue-specific promoter,    preferably to a PR promoter, most preferably to a PR promoter from    rice.-   11. Method according to any one of items 1 to 10, wherein said    nucleic acid encoding an ASPAT polypeptide is of plant origin,    preferably from a dicotyledonous plant, further preferably from the    family poaceae, more preferably from the genus Oryza, most    preferably from Oryza sativa.-   12. Plant or part thereof, including seeds, obtainable by a method    according to any one of items 1 to 11, wherein said plant or part    thereof comprises a recombinant nucleic acid encoding an ASPAT    polypeptide.-   13. Construct comprising:    -   (i) nucleic acid encoding an ASPAT polypeptide as defined in        items 1 or 2;    -   (ii) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (iii) a transcription termination sequence.-   14. Construct according to item 13, wherein one of said control    sequences is a constitutive promoter, preferably a GOS2 promoter,    most preferably a GOS2 promoter from rice.-   15. Construct according to item 13, wherein one of said control    sequences is a green tissue-specific promoter, preferably to a PR    promoter, most preferably to a PR promoter from rice.-   16. Use of a construct according to item 13 to 15 in a method for    making plants having increased yield, particularly increased biomass    and/or increased seed yield relative to control plants.-   17. Plant, plant part or plant cell transformed with a construct    according to item 13 to 15.-   18. Method for the production of a transgenic plant having increased    yield, particularly increased biomass and/or increased seed yield    relative to control plants, comprising:    -   (i) introducing and expressing in a plant a nucleic acid        encoding an ASPAT polypeptide as defined in item 1 or 2; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.-   19. Transgenic plant having increased yield, particularly increased    biomass and/or increased seed yield, relative to control plants,    resulting from modulated expression of a nucleic acid encoding an    ASPAT polypeptide as defined in item 1 or 2, or a transgenic plant    cell derived from said transgenic plant.-   20. Transgenic plant according to item 11, 17 or 18, or a transgenic    plant cell derived thereof, wherein said plant is a crop plant or a    monocot or a cereal, such as rice, maize, wheat, barley, millet,    rye, triticale, sorghum emmer, spelt, secale, einkorn, teff, milo    and oats.-   21. Harvestable parts of a plant according to item 20, wherein said    harvestable parts are preferably shoot biomass and/or seeds.-   22. Products derived from a plant according to item 20 and/or from    harvestable parts of a plant according to item 21.-   23. Use of a nucleic acid encoding an ASPAT polypeptide in    increasing yield, particularly in increasing seed yield and/or shoot    biomass in plants, relative to control plants.-   24. An isolated nucleic acid molecule selected from:    -   (a) a nucleic acid represented by any one of SEQ ID NO: 81, 147,        153, 183 and 185;    -   (b) the complement of a nucleic acid represented by any one of        SEQ ID NO: 81, 147, 153, 183 and 185;    -   (c) a nucleic acid encoding the polypeptide as represented by        any one of SEQ ID NO: 82, 148, 154, 184 and 186, preferably as a        result of the degeneracy of the genetic code, said isolated        nucleic acid can be derived from a polypeptide sequence as        represented by any one of SEQ ID NO: 82, 148, 154, 184 and 186        and further preferably confers enhanced yield-related traits        relative to control plants;    -   (d) a nucleic acid having, in increasing order of preference at        least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,        41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any        of the nucleic acid sequences of Table A1 and further preferably        conferring enhanced yield-related traits relative to control        plants;    -   (e) a nucleic acid molecule which hybridizes with a nucleic acid        molecule of (i) to (iv) under stringent hybridization conditions        and preferably confers enhanced yield-related traits relative to        control plants;    -   (f) a nucleic acid encoding an ASPAT polypeptide having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        amino acid sequence represented by any one of SEQ ID NO: 82,        148, 154, 184 and 186 and any of the other amino acid sequences        in Table A1 and preferably conferring enhanced yield-related        traits relative to control plants.-   25. An isolated polypeptide selected from:    -   (i) an amino acid sequence represented by any one of SEQ ID NO:        82, 148, 154, 184 and 186;    -   (ii) an amino acid sequence having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the amino acid sequence        represented by any one of SEQ ID NO: 82, 148, 154, 184 and 186,        and any of the other amino acid sequences in Table A and        preferably conferring enhanced yield-related traits relative to        control plants. (iii) derivatives of any of the amino acid        sequences given in (i) or (ii) above.        2. MYB91 like transcription factor (MYB91)-   1. A method for increasing yield-related traits in plants relative    to control plants, comprising increasing expression in a plant of a    nucleic acid sequence encoding a MYB91 like transcription factor    (MYB91) polypeptide, which MYB91 polypeptide comprises (i) (i) in    increasing order of preference at least 50%, 55%, 60%, 65%, 70%,    75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence    identity to a MYB DNA binding domain with an InterPro accession    number IPR014778, as represented by SEQ ID NO: 269; and (ii) in    increasing order of preference at least 50%, 55%, 60%, 65%, 70%,    75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence    identity to a MYB DNA binding domain with an InterPro accession    number IPR014778, as represented by SEQ ID NO: 270; and (iii) in    increasing order of preference at least 50%, 55%, 60%, 65%, 70%,    75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence    identity to a Conserved Domain as represented by SEQ ID NO: 271, and    optionally selecting for plants having increased yield-related    traits.-   2. Method according to item 1, wherein said MYB91 polypeptide    comprises in increasing order of preference at least 50%, 55%, 60%,    65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid    sequence identity to a polypeptide as represented by SEQ ID NO: 221.-   3. Method according to item 1, wherein said MYB91 polypeptide    comprises in increasing order of preference at least 50%, 55%, 60%,    65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid    sequence identity to any of the polypeptide sequences given in Table    A2 herein.-   4. Method according to item 1, wherein said MYB91 polypeptide, when    used in the construction of a phylogenetic tree of MYB DNA-binding    domain polypeptides, such as the one depicted in FIG. 4, clusters    with the MYB91 group of polypeptides rather than with any other    group.-   5. Method according to any preceding item, wherein said nucleic acid    sequence encoding a MYB91 polypeptide is represented by any one of    the nucleic acid sequence SEQ ID NOs given in Table A2 or a portion    thereof, or a sequence capable of hybridising with any one of the    nucleic acid sequences SEQ ID NOs given in Table A2, or to a    complement thereof.-   6. Method according to any preceding item, wherein said nucleic acid    sequence encodes an orthologue or paralogue of any of the    polypeptide sequence SEQ ID NOs given in Table A2.-   7. Method according to any preceding item, wherein said increased    expression is effected by any one or more of: T-DNA activation    tagging, TILLING, or homologous recombination.-   8. Method according to any preceding item, wherein said increased    expression is effected by introducing and expressing in a plant a    nucleic acid sequence encoding a MYB91 polypeptide.-   9. Method according to any preceding item, wherein said increased    yield-related trait is one or more of: increased plant height,    increased harvest index (HI), and/or increased Thousand Kernel    Weight (TKW).-   10. Method according to any preceding item, wherein said nucleic    acid sequence is operably linked to a constitutive promoter.-   11. Method according to item 10, wherein said constitutive promoter    is a GOS2 promoter, preferably a GOS2 promoter from rice, most    preferably a GOS2 sequence as represented by SEQ ID NO: 272.-   12. Method according to any preceding item, wherein said nucleic    acid sequence encoding a MYB91 polypeptide is from a plant, further    preferably from a dicotyledonous plant, more preferably from the    family Salicaceae, most preferably the nucleic acid sequence is from    Populus trichocarpa.-   13. Plants, parts thereof (including seeds), or plant cells    obtainable by a method according to any preceding item, wherein said    plant, part or cell thereof comprises an isolated nucleic acid    transgene encoding a MYB91 polypeptide.-   14. Construct comprising:    -   (a) a nucleic acid sequence encoding a MYB91 polypeptide as        defined in any one of items 1 to 6;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.-   15. Construct according to item 14 wherein said control sequence is    a constitutive promoter.-   16. Construct according to item 15 wherein said constitutive    promoter is a GOS2 promoter, preferably a GOS2 promoter from rice,    most preferably a GOS2 sequence as represented by SEQ ID NO: 272.-   17. Use of a construct according to any one of items 14 to 16 in a    method for making plants having increased yield-related traits    relative to control plants, which increased yield-related traits are    one or more of: increased plant height, increased harvest index    (HI), and increased Thousand Kernel Weight (TKW).-   18. Plant, plant part or plant cell transformed with a construct    according to any one of items 14 to 16.-   19. Method for the production of transgenic plants having increased    yield-related traits relative to control plants, comprising:    -   (i) introducing and expressing in a plant, plant part, or plant        cell, a nucleic acid sequence encoding a MYB91 polypeptide as        defined in any one of items 1 to 6; and    -   (ii) cultivating the plant cell, plant part, or plant under        conditions promoting plant growth and development.-   20. Transgenic plant having increased yield-related traits relative    to control plants, resulting from increased expression of an    isolated nucleic acid sequence encoding a MYB91 polypeptide as    defined in any one of items 1 to 6, or a transgenic plant cell or    transgenic plant part derived from said transgenic plant.-   21. Transgenic plant according to item 13, 18, or 20, wherein said    plant is a crop plant or a monocot or a cereal, such as rice, maize,    wheat, barley, millet, rye, triticale, sorghum and oats, or a    transgenic plant cell derived from said transgenic plant.-   22. Harvestable parts comprising an isolated nucleic acid sequence    encoding a MYB91 polypeptide, of a plant according to item 21,    wherein said harvestable parts are preferably seeds.-   23. Products derived from a plant according to item 21 and/or from    harvestable parts of a plant according to item 22.-   24. Use of a nucleic acid sequence encoding a MYB91 polypeptide as    defined in any one of items 1 to 6, in increasing yield-related    traits, comprising one or more of: increased plant height, increased    harvest index (HI), and increased Thousand Kernel Weight (TKW).

3. Gibberellic Acid-Stimulated Arabidopsis (GASA)

-   1. A method for enhancing yield-related traits in plants relative to    control plants, comprising modulating expression in a plant of a    nucleic acid encoding a GASA polypeptide, wherein the sequence of    said GASA polypeptide comprises a Pfam PF02704 domain, provided that    said GASA protein is not GASA4 as represented by SEQ ID NO: 295.-   2. Method according to item 1, wherein said GASA polypeptide    comprises one or more of the following motifs:

(b) Motif 4, (SEQ ID NO: 277) (c) Motif 5, (SEQ ID NO: 278) (d) Motif 6(SEQ ID NO: 279)

-   3. Method according to item 1 or 2, wherein said modulated    expression is effected by introducing and expressing in a plant a    nucleic acid encoding a GASA polypeptide.-   4. Method according to any one of items 1 to 3, wherein said nucleic    acid encoding a GASA polypeptide encodes any one of the proteins    listed in Table A3 or is a portion of such a nucleic acid, or a    nucleic acid capable of hybridising with such a nucleic acid.-   5. Method according to any one of items 1 to 4, wherein said nucleic    acid sequence encodes an orthologue or paralogue of any of the    proteins given in Table A3.-   6. Method according to any preceding item, wherein said enhanced    yield-related traits comprise increased seed yield relative to    control plants.-   7. Method according to any one of items 1 to 6, wherein said    enhanced yield-related traits are obtained under non-stress    conditions.-   8. Method according to any one of items 1 to 6, wherein said    enhanced yield-related traits are obtained under conditions of    drought stress, salt stress or nitrogen deficiency.-   9. Method according to any one of items 3 to 8, wherein said nucleic    acid is operably linked to a constitutive promoter, preferably to a    GOS2 promoter, most preferably to a GOS2 promoter from rice.-   10. Method according to any one of items 1 to 9, wherein said    nucleic acid encoding a GASA polypeptide is of plant origin,    preferably from a dicotyledonous plant.-   11. Plant or part thereof, including seeds, obtainable by a method    according to any one of items 1 to 10, wherein said plant or part    thereof comprises a recombinant nucleic acid encoding a GASA    polypeptide.-   12. Construct comprising:    -   (i) nucleic acid encoding a GASA polypeptide as defined in items        1 or 2;    -   (ii) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (iii) a transcription termination sequence.-   13. Construct according to item 12, wherein one of said control    sequences is a constitutive promoter, preferably a GOS2 promoter,    most preferably a GOS2 promoter from rice.-   14. Use of a construct according to item 12 or 13 in a method for    making plants having increased yield, particularly increased seed    yield relative to control plants.-   15. Plant, plant part or plant cell transformed with a construct    according to item 12 or 13.-   16. Method for the production of a transgenic plant having increased    yield, particularly increased seed yield relative to control plants,    comprising:    -   (i) introducing and expressing in a plant a nucleic acid        encoding a GASA polypeptide as defined in item 1 or 2; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.-   17. Transgenic plant having increased yield, particularly increased    seed yield, relative to control plants, resulting from modulated    expression of a nucleic acid encoding GASA polypeptide as defined in    item 1 or 2, or a transgenic plant cell derived from said transgenic    plant.-   18. Transgenic plant according to item 11, 15 or 17, or a transgenic    plant cell derived thereof, wherein said plant is a crop plant or a    monocot or a cereal, such as rice, maize, wheat, barley, millet,    rye, triticale, sorghum emmer, spelt, secale, einkorn, teff, milo    and oats.-   19. Harvestable parts of a plant according to item 18, wherein said    harvestable parts are seeds.-   20. Products derived from a plant according to item 18 and/or from    harvestable parts of a plant according to item 19.-   21. Use of a nucleic acid encoding a GASA polypeptide in increasing    yield, particularly in increasing seed yield in plants, relative to    control plants.

4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

-   1. A method for enhancing yield-related traits in plants relative to    control plants, comprising modulating expression in a plant of a    nucleic acid encoding an AUX/IAA polypeptide comprising an AUX/IAA    domain.-   2. Method according to item 1, wherein said AUX/IAA domain has in    increasing order of preference at least 25%, 26%, 27%, 28%, 29%,    30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,    43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,    56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,    69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,    82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid of an    AUX/IAA domain, preferably to the AUX/IAA domain of any of the    polypeptides of Table A4, most preferably to the AUX/IAA domain of    SEQ ID NO: 432 as represented by the amino acids located between    amino acid coordinates 5 to 171.-   3. Method according to item 1 wherein said AUX/IAA polypeptide is an    IAA14-like polypeptide comprises one or more of the following    motifs:    -   (i) Motif 13: SEQ ID NO: 739,    -   (ii) Motif 14: SEQ ID NO: 740,    -   (iii) Motif 15: SEQ ID NO: 741,    -   (iv) Motif 16: SEQ ID NO: 742,    -   (v) Motif 17: SEQ ID NO: 743,    -   (vi) Motif 18: SEQ ID NO: 744.-   4. Method according to item 1 to 3, wherein said modulated    expression is effected by introducing and expressing in a plant a    nucleic acid encoding an AUX/IAA polypeptide.-   5. Method according to any one of items 1 to 4, wherein said nucleic    acid encoding an AUX/IAA polypeptide encodes any one of the proteins    listed in Table A4 or in Table A5 or is a portion of such a nucleic    acid, or a nucleic acid capable of hybridising with such a nucleic    acid.-   6. Method according to any one of items 1 to 5, wherein said nucleic    acid sequence encodes an orthologue or paralogue of any of the    proteins given in Table A4 or in Table A5.-   7. Method according to any preceding item, wherein said enhanced    yield-related traits comprise increased yield, preferably increased    biomass and/or increased seed yield relative to control plants.-   8. Method according to any one of items 1 to 7, wherein said    enhanced yield-related traits are obtained under non-stress    conditions.-   9. Method according to any one of items 3 to 8, wherein said nucleic    acid is operably linked to a constitutive promoter, preferably to a    GOS2 promoter, most preferably to a GOS2 promoter from rice.-   10. Method according to any one of items 1 to 9, wherein said    nucleic acid encoding an AUX/IAA polypeptide is of plant origin,    preferably from a monocotyledonous plant, further preferably from    the family Poaceae, more preferably from the genus Oryza, most    preferably from Oryza sativa.-   11. Plant or part thereof, including seeds, obtainable by a method    according to any one of items 1 to 10, wherein said plant or part    thereof comprises a recombinant nucleic acid encoding an AUX/IAA    polypeptide.-   12. Construct comprising:    -   (i) nucleic acid encoding an AUX/IAA polypeptide as defined in        items 1 or 2;    -   (ii) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (iii) a transcription termination sequence.-   13. Construct according to item 12, wherein one of said control    sequences is a constitutive promoter, preferably a GOS2 promoter,    most preferably a GOS2 promoter from rice.-   14. Use of a construct according to item 12 or 13 in a method for    making plants having increased yield, particularly increased biomass    and/or increased seed yield relative to control plants.-   15. Plant, plant part or plant cell transformed with a construct    according to item 12 or 13.-   16. Method for the production of a transgenic plant having increased    yield, particularly increased biomass and/or increased seed yield    relative to control plants, comprising:    -   (i) introducing and expressing in a plant a nucleic acid        encoding an AUX/IAA polypeptide as defined in item 1 or 2; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.-   17. Transgenic plant having increased yield, particularly increased    biomass and/or increased seed yield, relative to control plants,    resulting from modulated expression of a nucleic acid encoding an    AUX/IAA polypeptide as defined in item 1 or 2, or a transgenic plant    cell derived from said transgenic plant.-   18. Transgenic plant according to item 11, 15 or 17, or a transgenic    plant cell derived thereof, wherein said plant is a crop plant or a    monocot or a cereal, such as rice, maize, wheat, barley, millet,    rye, triticale, sorghum emmer, spelt, secale, einkorn, teff, milo    and oats.-   19. Harvestable parts of a plant according to item 18, wherein said    harvestable parts are preferably shoot biomass and/or seeds.-   20. Products derived from a plant according to item 18 and/or from    harvestable parts of a plant according to item 19.-   21. Use of a nucleic acid encoding an AUX/IAA polypeptide in    increasing yield, particularly in increasing seed yield and/or shoot    biomass in plants, relative to control plants.

DESCRIPTION OF FIGURES

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 represents a multiple alignment of ASPAT polypeptides.

FIG. 2 shows a phylogenetic tree of ASPAT polypeptides.

FIG. 3 represents the binary vector used for increased expression inOryza sativa of an ASPAT-encoding nucleic acid under the control of arice GOS2 promoter (pGOS2) or of a rice PR promoter.

FIG. 4 represents the phylogenetic relationship among MYB DNA bindingdomain polypeptides from Arabidopsis thaliana and from other plants,based upon amino acid sequence (according to Stracke et al. (2004)Current Opinion in Plant Biology 2001, 4:447-456). The MYB polypeptideswere clustered using PHYLIP, and motifs were detected using MEME.Polypeptides useful in performing the methods of the invention clusterwith MYB91, circled and marked by a black arrow.

FIG. 5 shows a ClustalW 1.81 multiple sequence alignment of the MYB91polypeptides from Table A2. Two MYB DNA binding domains with an InterProaccession number IPR014778, a MYB transcription factor with an InterProaccession number IPR015495, and a C-terminal Conserved Domain, aremarked with X's below the consensus sequence.

FIG. 6 shows the binary vector for increased expression in Oryza sativaplants of a nucleic acid sequence encoding a MYB91 polypeptide under thecontrol of a promoter functioning in plants.

FIG. 7 represents the domain structure of SEQ ID NO: 276 with the GASAdomain PF02704 indicated in bold. The putative secretion signal peptide(amino acid 1-24) is underlined.

FIG. 8 represents a multiple alignment of various GASA proteins. Themotifs 4 to 12 or other motifs can be deduced herefrom.

FIG. 9 shows a phylogenetic tree of Arabidopsis GASA proteins (Roxrud etal. 2007). Starting from a multiple alignment with ClustalW (Thompson etal., Nucleic Acids Res. 22, 4673-4680, 1994), a neighbour-joiningphylogenetic tree was obtained using the PAUP v.4.0 software(http://www.paup. csit.fsu.edu), and statistical confidence wascalculated by bootstrap analysis with 1,000 resamplings.

FIG. 10 represents the binary vector for increased expression in Oryzasativa of a GASA-encoding nucleic acid under the control of a rice GOS2promoter (pGOS2).

FIG. 11 represents a multiple alignment of AUX/IAA polypeptides.

FIG. 12 represents the binary vector used for increased expression inOryza sativa of an AUX/IAA encoding nucleic acid under the control of arice GOS2 promoter (pGOS2).

FIG. 13 represents the domain structure of SEQ ID NO: 738 with theAUX/IAA domain in bold and the conserved motifs underlined.

FIG. 14 represents a multiple alignment of IAA14-like protein sequences.

FIG. 15 shows a neighbour-joining tree of Arabidopsis IAA proteins(Remington et al., 2004). SEQ ID NO: 738 is represented by IAA14 inGroup A and IAA14-like proteins preferably cluster in this Group A.

FIG. 16 represents the binary vector used for increased expression inOryza sativa of an IAA14-like-encoding nucleic acid under the control ofa rice HMGP promoter (pHMGP).

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone. Thefollowing examples are not intended to completely define or otherwiselimit the scope of the invention.

DNA manipulation: unless otherwise stated, recombinant DNA techniquesare performed according to standard protocols described in (Sambrook(2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold SpringHarbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubelet al. (1994), Current Protocols in Molecular Biology, CurrentProtocols. Standard materials and methods for plant molecular work aredescribed in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications (UK).

Example 1 Identification of Sequences Related to the Nucleic AcidSequence Used in the Methods of the Invention

Sequences (full length cDNA, ESTs or genomic) related to the nucleicacid sequence used in the methods of the present invention wereidentified amongst those maintained in the Entrez Nucleotides databaseat the National Center for Biotechnology Information (NCBI) usingdatabase sequence search tools, such as the Basic Local Alignment Tool(BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used tofind regions of local similarity between sequences by comparing nucleicacid or polypeptide sequences to sequence databases and by calculatingthe statistical significance of matches. For example, the polypeptideencoded by the nucleic acid used in the present invention was used forthe TBLASTN algorithm, with default settings and the filter to ignorelow complexity sequences set off. The output of the analysis was viewedby pairwise comparison, and ranked according to the probability score(E-value), where the score reflect the probability that a particularalignment occurs by chance (the lower the E-value, the more significantthe hit). In addition to E-values, comparisons were also scored bypercentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In someinstances, the default parameters may be adjusted to modify thestringency of the search. For example the E-value may be increased toshow less stringent matches. This way, short nearly exact matches may beidentified.

1.1. Aspartate AminoTransferase (ASPAT)

Table A1 provides a list of nucleic acid sequences related to thenucleic acid sequence used in the methods of the present invention.

TABLE A1 Examples of ASPAT polypeptides: Amino acid Reference Nucleicacid SEQ ID number Name SEQ ID NO: NO: 1 O. sativa_Os01g0760600 1 2 1 O.sativa_Os01g0760600- 3 4 truncated 1 A. thaliana_AT5G19550 5 6 1 A.thaliana_AT5G11520 7 8 1 A. thaliana_AT4G31990 9 10 6 A.thaliana_AT1G62800 11 12 7 B. napus_TA23207 13 14 8 B. napus_TA23768 1516 9 C. sinensis_TA12564 17 18 10 C. solstitialis_TA659 19 20 11 G.hirsutum_TA23799 21 22 12 G. max_AF034210 23 24 13 G. raimondii_TA941325 26 14 H. annuus_TA8926 27 28 15 H. paradoxus_TA2606 29 30 16 J.regia_TA762 31 32 17 L. japonicus_TA1537 33 34 18 L. perennis_TA512 3536 19 L. perennis_TA605 37 38 20 N. tabacum_TA13125 39 40 21 P.glauca_TA15326 41 42 22 P. patens_136815 43 44 23 P. persica_TA3273 4546 24 P. sitchensis_TA22265 47 48 25 P. trichocarpa_819551 49 50 26 P.trifoliata_TA8305 51 52 27 S. lycopersicum_TA38054 53 54 28 S.officinarum_TA26595 55 56 29 T. aestivum_TA52678 57 58 30 V.carteri_82929 59 60 31 V. vinifera_GSVIVT00016723001 61 62 32 V.vinifera_GSVIVT00032463001 63 64 33 Z. mays_TA9042 65 66 34 C.reinhardtii_186959 67 68 35 C. solstitialis_TA2275 69 70 36 C.tinctorius_TA12 71 72 37 G. hirsutum_TA24406 73 74 38 G. max_TA61768 7576 39 G. raimondii_TA9928 77 78 40 H. exilis_TA1663 79 80 41 H.vulgare_BPS_7992 81 82 42 L. japonicus_TA1466 83 84 43 M.polymorpha_TA825 85 86 44 N. tabacum_TA13015 87 88 45 O.sativa_Os02g0797500 89 90 46 P. glauca_TA14780 91 92 47 P. patens_10213493 94 48 P. sitchensis_TA20968 95 96 49 P. taeda_TA6616 97 98 50 P.trichocarpa_654206 99 100 51 P. trichocarpa_835828 101 102 52 P.vulgaris_TA4043 103 104 53 S. tuberosum_TA23192 105 106 54 V.carteri_81153 107 108 55 V. vinifera_GSVIVT00032723001 109 110 56 Z.mays_TA10886 111 112 57 A. thaliana_AT2G30970 113 114 58 C.sinensis_TA15250 115 116 59 G. max_TA50178 117 118 60 G.raimondii_TA9985 119 120 61 H. vulgare_TA32835 121 122 62 H.vulgare_TA36301 123 124 63 O. lucimarinus_31597 125 126 64 O.sativa_Os02g0236000 127 128 65 O. sativa_Os06g0548000 129 130 66 O.taurii_32764 131 132 67 P. patens_169868 133 134 68 P.sitchensis_TA23007 135 136 69 P. taeda_TA7145 137 138 70 V.vinifera_GSVIVT00018772001 139 140 71 V. vinifera_GSVIVT00037462001 141142 72 A. anophagefferens_21970 143 144 73 A. thaliana_AT2G22250.2 145146 74 B. napus_BPS_9867 147 148 75 C. reinhardtii_118364 149 150 76 G.hirsutum_TA27281 151 152 77 G. max_BPS_36342 153 154 78 H.vulgare_TA28738 155 156 79 M. domestica_TA26867 157 158 80 N.tabacum_TA15308 159 160 81 O. basilicum_TA1043 161 162 82 O.sativa_Os01g0871300 163 164 83 P. patens_127152 165 166 84 P.pinaster_TA3616_71647 167 168 85 P. trichocarpa_scaff_V.183 169 170 86P. trichocarpa_scaff_VII.574 171 172 87 S. lycopersicum_TA37592 173 17488 S. tuberosum_TA27739 175 176 89 T. aestivum_TA71539 177 178 90 V.carteri_103084 179 180 91 V. vinifera_GSVIVT00019453001 181 182 92 Z.mays_BPS_26636 183 184 93 Z. mays_BPS_4233 185 186

In some instances, related sequences is tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR; beginning with TA). The Eukaryotic GeneOrthologs (EGO) database may be used to identify such related sequences,either by keyword search or by using the BLAST algorithm with thenucleic acid sequence or polypeptide sequence of interest. On otherinstances, special nucleic acid sequence databases have been fromparticular organisms, such as those maintained by the Joint GenomeInstitute, like the poplar genome sequences have been screened.

Further, access to proprietary databases, has allowed the identificationof other nucleic acid and polypeptide sequences using the Blastalgorithm as described above.

1.2. MYB91 like transcription factor (MYB91)

Table A2 provides a list of nucleic acid sequences related to thenucleic acid sequence used in the methods of the present invention.

TABLE A2 Examples of MYB91 polypeptide sequences, and encoding nucleicacid sequences Nucleic Poly- acid peptide Public database SEQ ID SEQ IDName accession number NO: NO: Poptr_MYB91 NA 220 221 Antma_MYB91AJ005586 222 223 (PHAN) Aqufo_MYB91 DR919410 224 225 DR919310Arath_MYB91 (AS1) AT2G37630 226 227 Brana_MYB91 BN06MC30974_51405116 228229 @30844#1 Carhi_MYB91 DQ512733 230 231 Escca_MYB91 AY228766 232 233Eucgr_MYB91 BD376532 234 235 Glyma_MYB91 AY790252 236 237 (PHANa)Glyma_MYB91 AY790253 238 239 (PHANb) Goshi_MYB91 DT554770 240 241DW499296 Lotco_MYB91 AY790244 242 243 (PHANa) Lotco_MYB91 AY790245 244245 (PHANb) Lyces_MYB91 AF148934 246 247 Maldo_MYB91 DQ074473 248 249Medtr_MYB91 DQ468322 250 251 PHAN Moral_MYB91 EF408927 252 253 PHAN1Nicta_MYB91 AY559043 254 255 Orysa_MYB91 Os12g0572000 256 257NM_001073621 Pissa_MYB91 AF299140.2 258 259 (PHAN1) Soltu_MYB91 CK274535260 261 Vitvi_MYB91 AM474349 262 263 Zeama_MYB91 AF126489 264 265 (RS2)Horvu_MYB91 BF617675.2 266 267 partial BG343686.1

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR; beginning with TA). The Eukaryotic GeneOrthologs (EGO) database may be used to identify such related sequences,either by keyword search or by using the BLAST algorithm with thenucleic acid sequence or polypeptide sequence of interest. On otherinstances, special nucleic acid sequence databases have been created forparticular organisms, such as by the Joint Genome Institute. Further,access to proprietary databases, has allowed the identification of novelnucleic acid and polypeptide sequences.

1.3. Gibberellic Acid-Stimulated Arabidopsis (GASA) Table A3 provides alist of nucleic acid sequences related to the nucleic acid sequence usedin the methods of the present invention.

TABLE A3 Examples of GASA polypeptides: Polypeptide Nucleic acid NameSEQ ID NO SEQ ID NO Le_GASA growth induced 276 275 Pop_GASA growthregulated 291 361 Mt_GASA growth regulated 292 362 GASA12 At2g30810 293363 GASA5 At3g02885 294 364 GASA4 At5g15230 295 365 GASA6 At1g74670 296366 TA5035_4679#1 297 367 TA5923_4679#1 298 368 TA3842_4679#1 299 369Os05g0376800#1 300 370 Os04g0465300#1 301 371 Os10g0115550#1 302 372AK105729#1 303 373 Os05g0432200#1 304 374 Os09g0414900#1 305 375Os03g0607200#1 306 376 Os07g0592000#1 307 377 AK110640#1 308 378Os06g0266800#1 309 379 Os03g0760800#1 310 380 scaff_205.30#1 311 381scaff_II.204#1 312 382 scaff_II.2330#1 313 383 scaff_IX.735#1 314 384scaff_VI.397#1 315 385 scaff_XVII.377#1 316 386 scaff_II.202#1 317 387scaff_I.2410#1 318 388 scaff_I.1483#1 319 389 scaff_I.1926#1 320 390scaff_XII.704#1 321 391 scaff_40.379#1 322 392 scaff_41.75#1 323 393scaff_XV.507#1 324 394 scaff_II.2328#1 325 395 scaff_II.203#1 326 396scaff_XIX.758#1 327 397 TA45751_4081#1 328 398 TA48119_4081#1 329 399TA35962_4081#1 330 400 BI208422#1 331 401 BG128975#1 332 402TA52374_4081#1 333 403 TA37180_4081#1 334 404 BE353147#1 335 405TA56938_4081#1 336 406 BG130916#1 337 407 TA52635_4081#1 338 408TA41886_4081#1 339 409 TA36295_4081#1 340 410 TA56201_4081#1 341 411AJ785329#1 342 412 CA725087#1 343 413 TA69823_4565#1 344 414TA53297_4565#1 345 415 TA101332_4565#1 346 416 TA66036_4565#1 347 417TA100367_4565#1 348 418 TA92393_4565#1 349 419 BM136027#1 350 420CA705831#1 351 421 CA593033#1 352 422 CK153563#1 353 423 TA66038_4565#1354 424 TA52915_4565#1 355 425 TA69821_4565#1 356 426 TA95153_4565#1 357427 CD899399#1 358 428 TA77646_4565#1 359 429 TA51752_4565#1 360 430

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) databasemay be used to identify such related sequences, either by keyword searchor by using the BLAST algorithm with the nucleic acid or polypeptidesequence of interest.

1.4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

Nucleic Acid Polypeptide Nucleic acid name SEQ ID NO: Polypeptide nameSEQ ID NO: seqidno01; DNA; Oryza sativa 431 seqidno02; PRT; Oryza sativa432 seqidno1; DNA; Arabidopsis thaliana 433 seqidno2; PRT; Arabidopsisthaliana 434 seqidno3; DNA; Arabidopsis thaliana 435 seqidno4; PRT;Arabidopsis thaliana 436 seqidno5; DNA; Arabidopsis thaliana 437seqidno6; PRT; Arabidopsis thaliana 438 seqidno7; DNA; Arabidopsisthaliana 439 seqidno8; PRT; Arabidopsis thaliana 440 seqidno9; DNA;Arabidopsis thaliana 441 seqidno10; PRT; Arabidopsis thaliana 442seqidno11; DNA; Arabidopsis thaliana 443 seqidno12; PRT; Arabidopsisthaliana 444 seqidno13; DNA; Arabidopsis thaliana 445 seqidno14; PRT;Arabidopsis thaliana 446 seqidno15; DNA; Arabidopsis thaliana 447seqidno16; PRT; Arabidopsis thaliana 448 seqidno17; DNA; Arabidopsisthaliana 449 seqidno18; PRT; Arabidopsis thaliana 450 seqidno19; DNA;Arabidopsis thaliana 451 seqidno20; PRT; Arabidopsis thaliana 452seqidno21; DNA; Arabidopsis thaliana 453 seqidno22; PRT; Arabidopsisthaliana 454 seqidno23; DNA; Arabidopsis thaliana 455 seqidno24; PRT;Arabidopsis thaliana 456 seqidno25; DNA; Arabidopsis thaliana 457seqidno26; PRT; Arabidopsis thaliana 458 seqidno27; DNA; Arabidopsisthaliana 459 seqidno28; PRT; Arabidopsis thaliana 460 seqidno29; DNA;Arabidopsis thaliana 461 seqidno30; PRT; Arabidopsis thaliana 462seqidno31; DNA; Arabidopsis thaliana 463 seqidno32; PRT; Arabidopsisthaliana 464 seqidno33; DNA; Arabidopsis thaliana 465 seqidno34; PRT;Arabidopsis thaliana 466 seqidno35; DNA; Arabidopsis thaliana 467seqidno36; PRT; Arabidopsis thaliana 468 seqidno37; DNA; Arabidopsisthaliana 469 seqidno38; PRT; Arabidopsis thaliana 470 seqidno39; DNA;Arabidopsis thaliana 471 seqidno40; PRT; Arabidopsis thaliana 472seqidno41; DNA; Arabidopsis thaliana 473 seqidno42; PRT; Arabidopsisthaliana 474 seqidno43; DNA; Arabidopsis thaliana 475 seqidno44; PRT;Arabidopsis thaliana 476 seqidno45; DNA; Arabidopsis thaliana 477seqidno46; PRT; Arabidopsis thaliana 478 seqidno47; DNA; Arabidopsisthaliana 479 seqidno48; PRT; Arabidopsis thaliana 480 seqidno49; DNA;Arabidopsis thaliana 481 seqidno50; PRT; Arabidopsis thaliana 482seqidno51; DNA; Arabidopsis thaliana 483 seqidno52; PRT; Arabidopsisthaliana 484 seqidno53; DNA; Arabidopsis thaliana 485 seqidno54; PRT;Arabidopsis thaliana 486 seqidno55; DNA; Arabidopsis thaliana 487seqidno56; PRT; Arabidopsis thaliana 488 seqidno57; DNA; Arabidopsisthaliana 489 seqidno58; PRT; Arabidopsis thaliana 490 seqidno59; DNA;Arabidopsis thaliana 491 seqidno60; PRT; Arabidopsis thaliana 492seqidno61; DNA; Arabidopsis thaliana 493 seqidno62; PRT; Arabidopsisthaliana 494 seqidno63; DNA; Arabidopsis thaliana 495 seqidno64; PRT;Arabidopsis thaliana 496 seqidno65; DNA; Arabidopsis thaliana 497seqidno66; PRT; Arabidopsis thaliana 498 seqidno67; DNA; Arabidopsisthaliana 499 seqidno68; PRT; Arabidopsis thaliana 500 seqidno69; DNA;Oryza sativa 501 seqidno70; PRT; Oryza sativa 502 seqidno71; DNA; Oryzasativa 503 seqidno72; PRT; Oryza sativa 504 seqidno73; DNA; Oryza sativa505 seqidno74; PRT; Oryza sativa 506 seqidno75; DNA; Oryza sativa 507seqidno76; PRT; Oryza sativa 508 seqidno77; DNA; Oryza sativa 509seqidno78; PRT; Oryza sativa 510 seqidno79; DNA; Oryza sativa 511seqidno80; PRT; Oryza sativa 512 seqidno81; DNA; Oryza sativa 513seqidno82; PRT; Oryza sativa 514 seqidno83; DNA; Oryza sativa 515seqidno84; PRT; Oryza sativa 516 seqidno85; DNA; Oryza sativa 517seqidno86; PRT; Oryza sativa 518 seqidno87; DNA; Oryza sativa 519seqidno88; PRT; Oryza sativa 520 seqidno89; DNA; Oryza sativa 521seqidno90; PRT; Oryza sativa 522 seqidno91; DNA; Oryza sativa 523seqidno92; PRT; Oryza sativa 524 seqidno93; DNA; Oryza sativa 525seqidno94; PRT; Oryza sativa 526 seqidno95; DNA; Oryza sativa 527seqidno96; PRT; Oryza sativa 528 seqidno97; DNA; Oryza sativa 529seqidno98; PRT; Oryza sativa 530 seqidno99; DNA; Oryza sativa 531seqidno100; PRT; Oryza sativa 532 seqidno101; DNA; Oryza sativa 533seqidno102; PRT; Oryza sativa 534 seqidno103; DNA; Oryza sativa 535seqidno104; PRT; Oryza sativa 536 seqidno105; DNA; Oryza sativa 537seqidno106; PRT; Oryza sativa 538 seqidno107; DNA; Oryza sativa 539seqidno108; PRT; Oryza sativa 540 seqidno109; DNA; Oryza sativa 541seqidno110; PRT; Oryza sativa 542 seqidno111; DNA; Oryza sativa 543seqidno112; PRT; Oryza sativa 544 seqidno113; DNA; Oryza sativa 545seqidno114; PRT; Oryza sativa 546 seqidno115; DNA; Oryza sativa 547seqidno116; PRT; Oryza sativa 548 seqidno117; DNA; Oryza sativa 549seqidno118; PRT; Oryza sativa 550 seqidno119; DNA; Oryza sativa 551seqidno120; PRT; Oryza sativa 552 seqidno121; DNA; Oryza sativa 553seqidno122; PRT; Oryza sativa 554 seqidno123; DNA; Oryza sativa 555seqidno124; PRT; Oryza sativa 556 seqidno125; DNA; Oryza sativa 557seqidno126; PRT; Oryza sativa 558 seqidno127; DNA; Oryza sativa 559seqidno128; PRT; Oryza sativa 560 seqidno129; DNA; Oryza sativa 561seqidno130; PRT; Oryza sativa 562 seqidno131; DNA; Oryza sativa 563seqidno132; PRT; Oryza sativa 564 seqidno133; DNA; Oryza sativa 565seqidno134; PRT; Oryza sativa 566 seqidno135; DNA; Oryza sativa 567seqidno136; PRT; Oryza sativa 568 seqidno137; DNA; Oryza sativa 569seqidno138; PRT; Oryza sativa 570 seqidno139; DNA; Oryza sativa 571seqidno140; PRT; Oryza sativa 572 seqidno141; DNA; Oryza sativa 573seqidno142; PRT; Oryza sativa 574 seqidno143; DNA; Oryza sativa 575seqidno144; PRT; Oryza sativa 576 seqidno145; DNA; Oryza sativa 577seqidno146; PRT; Oryza sativa 578 seqidno147; DNA; Oryza sativa 579seqidno148; PRT; Oryza sativa 580 seqidno149; DNA; Oryza sativa 581seqidno150; PRT; Oryza sativa 582 seqidno151; DNA; Oryza sativa 583seqidno152; PRT; Oryza sativa 584 seqidno153; DNA; Oryza sativa 585seqidno154; PRT; Oryza sativa 586 seqidno155; DNA; Oryza sativa 587seqidno156; PRT; Oryza sativa 588 seqidno157; DNA; Oryza sativa 589seqidno158; PRT; Oryza sativa 590 seqidno159; DNA; Oryza sativa 591seqidno160; PRT; Oryza sativa 592 seqidno161; DNA; Oryza sativa 593seqidno162; PRT; Oryza sativa 594 seqidno163; DNA; Oryza sativa 595seqidno164; PRT; Oryza sativa 596 seqidno165; DNA; Oryza sativa 597seqidno166; PRT; Oryza sativa 598 seqidno167; DNA; Oryza sativa 599seqidno168; PRT; Oryza sativa 600 seqidno169; DNA; Oryza sativa 601seqidno170; PRT; Oryza sativa 602 seqidno171; DNA; Oryza sativa 603seqidno172; PRT; Oryza sativa 604 seqidno173; DNA; Oryza sativa 605seqidno174; PRT; Oryza sativa 606 seqidno175; DNA; Oryza sativa 607seqidno176; PRT; Oryza sativa 608 seqidno177; DNA; Oryza sativa 609seqidno178; PRT; Oryza sativa 610 seqidno179; DNA; Oryza sativa 611seqidno180; PRT; Oryza sativa 612 seqidno181; DNA; Oryza sativa 613seqidno182; PRT; Oryza sativa 614 seqidno183; DNA; Oryza sativa 615seqidno184; PRT; Oryza sativa 616 seqidno185; DNA; Oryza sativa 617seqidno186; PRT; Oryza sativa 618 seqidno187; DNA; Oryza sativa 619seqidno188; PRT; Oryza sativa 620 seqidno189; DNA; Oryza sativa 621seqidno190; PRT; Oryza sativa 622 seqidno191; DNA; Oryza sativa 623seqidno192; PRT; Oryza sativa 624 seqidno193; DNA; Oryza sativa 625seqidno194; PRT; Oryza sativa 626 seqidno195; DNA; Zea mays 627seqidno196; PRT; Zea mays 628 seqidno197; DNA; Zea mays 629 seqidno198;PRT; Zea mays 630 seqidno199; DNA; Zea mays 631 seqidno200; PRT; Zeamays 632 seqidno201; DNA; Zea mays 633 seqidno202; PRT; Zea mays 634seqidno203; DNA; Zea mays 635 seqidno204; PRT; Zea mays 636 seqidno205;DNA; Zea mays 637 seqidno206; PRT; Zea mays 638 seqidno207; DNA; Zeamays 639 seqidno208; PRT; Zea mays 640 seqidno209; DNA; Zea mays 641seqidno210; PRT; Zea mays 642 seqidno211; DNA; Zea mays 643 seqidno212;PRT; Zea mays 644 seqidno213; DNA; Zea mays 645 seqidno214; PRT; Zeamays 646 seqidno215; DNA; Zea mays 647 seqidno216; PRT; Zea mays 648seqidno217; DNA; Zea mays 649 seqidno218; PRT; Zea mays 650 seqidno219;DNA; Zea mays 651 seqidno220; PRT; Zea mays 652 seqidno221; DNA; Zeamays 653 seqidno222; PRT; Zea mays 654 seqidno223; DNA; Zea mays 655seqidno224; PRT; Zea mays 656 seqidno225; DNA; Zea mays 657 seqidno226;PRT; Zea mays 658 seqidno227; DNA; Zea mays 659 seqidno228; PRT; Zeamays 660 seqidno229; DNA; Zea mays 661 seqidno230; PRT; Zea mays 662seqidno231; DNA; Zea mays 663 seqidno232; PRT; Zea mays 664 seqidno233;DNA; Zea mays 665 seqidno234; PRT; Zea mays 666 seqidno673; DNA; Populustrichocarpa 673 seqidno674; PRT; Populus trichocarpa 674 seqidno675;DNA; Populus trichocarpa 675 seqidno676; PRT; Populus trichocarpa 676seqidno677; DNA; Populus trichocarpa 677 seqidno678; PRT; Populustrichocarpa 678 seqidno679; DNA; Populus trichocarpa 679 seqidno680;PRT; Populus trichocarpa 680 seqidno681; DNA; Populus trichocarpa 681seqidno682; PRT; Populus trichocarpa 682 seqidno683; DNA; Populustrichocarpa 683 seqidno684; PRT; Populus trichocarpa 684 seqidno685;DNA; Populus trichocarpa 685 seqidno686; PRT; Populus trichocarpa 686seqidno687; DNA; Populus trichocarpa 687 seqidno688; PRT; Populustrichocarpa 688 seqidno689; DNA; Populus trichocarpa 689 seqidno690;PRT; Populus trichocarpa 690 seqidno691; DNA; Populus trichocarpa 691seqidno692; PRT; Populus trichocarpa 692 seqidno693; DNA; Populustrichocarpa 693 seqidno694; PRT; Populus trichocarpa 694 seqidno695;DNA; Populus trichocarpa 695 seqidno696; PRT; Populus trichocarpa 696seqidno697; DNA; Populus trichocarpa 697 seqidno698; PRT; Populustrichocarpa 698 seqidno699; DNA; Populus trichocarpa 699 seqidno700;PRT; Populus trichocarpa 700 seqidno701; DNA; Populus trichocarpa 701seqidno702; PRT; Populus trichocarpa 702 seqidno703; DNA; Populustrichocarpa 703 seqidno704; PRT; Populus trichocarpa 704 seqidno705;DNA; Populus trichocarpa 705 seqidno706; PRT; Populus trichocarpa 706seqidno707; DNA; Populus trichocarpa 707 seqidno708; PRT; Populustrichocarpa 708 seqidno709; DNA; Populus trichocarpa 709 seqidno710;PRT; Populus trichocarpa 710 seqidno711; DNA; Populus trichocarpa 711seqidno712; PRT; Populus trichocarpa 712 seqidno713; DNA; Populustrichocarpa 713 seqidno714; PRT; Populus trichocarpa 714 seqidno715;DNA; Populus trichocarpa 715 seqidno716; PRT; Populus trichocarpa 716seqidno717; DNA; Populus trichocarpa 717 seqidno718; PRT; Populustrichocarpa 718 seqidno719; DNA; Populus trichocarpa 719 seqidno720;PRT; Populus trichocarpa 720 seqidno721; DNA; Populus trichocarpa 721seqidno722; PRT; Populus trichocarpa 722 seqidno723; DNA; Populustrichocarpa 723 seqidno724; PRT; Populus trichocarpa 724 seqidno725;DNA; Populus trichocarpa 725 seqidno726; PRT; Populus trichocarpa 726seqidno727; DNA; Populus trichocarpa 727 seqidno728; PRT; Populustrichocarpa 728 seqidno729; DNA; Populus trichocarpa 729 seqidno730;PRT; Populus trichocarpa 730 seqidno731; DNA; Populus trichocarpa 731seqidno732; PRT; Populus trichocarpa 732 seqidno733; DNA; Populustrichocarpa 733 seqidno734; PRT; Populus trichocarpa 734 seqidno735;DNA; Populus trichocarpa 735 seqidno736; PRT; Populus trichocarpa 736

1.5. IAA14 Polypeptides

Table A5 provides a list of nucleic acid sequences related to thenucleic acid sequence used in the methods of the present invention.

TABLE A5 Examples of IAA14-like polypeptides: Polypeptide Nucleic acidPlant Source Name SEQ ID NO: SEQ ID NO: Arabidopsis thalianaAT4G14550.1#1 738 737 Arabidopsis thaliana AT3G23050.1#1 748 783Arabidopsis thaliana AT3G23050.2#1 749 784 Populus trichocarpa 566151#1750 785 Populus trichocarpa 720961#1 751 786 Medicago truncatulaTA20354_3880#1 752 787 Solanum lycopersicum TA40922_4081#1 753 788Arabidopsis thaliana AT1G04250.1#1 754 789 Oryza sativa CB657009#1 755790 Oryza sativa TA41733_4530#1 756 791 Medicago truncatulaTA20951_3880#1 757 792 Arabidopsis thaliana AT3G04730.1#1 758 793Solanum lycopersicum TA48108_4081#1 759 794 Medicago truncatulaTA27011_3880#1 760 795 Medicago truncatula TA22814_3880#1 761 796Populus trichocarpa 643213#1 762 797 Arabidopsis thaliana AT3G23030.1#1763 798 Arabidopsis thaliana AT4G14560.1#1 764 799 Arabidopsis thalianaAT1G04240.1#1 765 800 Solanum lycopersicum TA38817_4081#1 766 801Solanum lycopersicum TA43058_4081#1 767 802 Populus trichocarpa 726443#1768 803 Populus trichocarpa 564913#1 769 804 Populus trichocarpa831610#1 770 805 Populus trichocarpa 798526#1 771 806 Medicagotruncatula TA20557_3880#1 772 807 Medicago truncatula TA20558_3880#1 773808 Populus trichocarpa 823671#1 774 809 Populus trichocarpa 595419#1775 810 Medicago truncatula TA31746_3880#1 776 811 Solanum lycopersicumTA42190_4081#1 777 812 Arabidopsis thaliana AT4G29080.1#1 778 813Medicago truncatula TA25400_3880#1 779 814 Populus trichocarpa 711734#1780 815 Populus trichocarpa 584053#1 781 816 Medicago truncatulaTA23062_3880#1 782 817

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) databasemay be used to identify such related sequences, either by keyword searchor by using the BLAST algorithm with the nucleic acid or polypeptidesequence of interest.

Example 2 Alignment of Sequences Related to the Polypeptide SequencesUsed in the Methods of the Invention 2.1. Aspartate AminoTransferase(ASPAT)

Alignment of polypeptide sequences was performed using the ClustalW 2.0algorithm of progressive alignment (Thompson et al. (1997) Nucleic AcidsRes 25:4876-4882; Chema et al. (2003). Nucleic Acids Res 31:3497-3500)with standard setting (slow alignment, similarity matrix: Gonnet (orBlosum 62 (if polypeptides are aligned), gap opening penalty 10, gapextension penalty: 0.2). Minor manual editing was done to furtheroptimise the alignment. The ASPAT polypeptides are aligned in FIG. 1.

A phylogenetic tree of ASPAT polypeptides (FIG. 2) was constructed usinga neighbour-joining clustering algorithm as provided in the AlignXprogramme from the Vector NTI (Invitrogen). The polypeptides clusteredin five major phylognetic classes, class 1, class 2, class 3, class 4,and class 5. Table B1 shows the polypeptides found within each of thefive classes. The polypeptides of Class 5 were used as an outgroup inthe phylogenectic analysis and do not represent ASPAT polypeptides.Therefore polypeptides of Class 5 are not part of the invention hereindescribed. Polypeptides within class 1 and 2 are typically expressed inthe cytosol or the chloroplast. Class 5 corresponds to the new class ofASAPT polypeptides defined by De La Torre et al. 2006. Polypeptideswithin class 4 are typically expressed in the mitochondria.

TABLE B1 Phylogenetic classes of ASPAT polypeptides. Phylo- Nucleic acidAmino acid genetic Name SEQ ID NO: SEQ ID NO: class O.sativa_Os01g0760600 1 2 1 O. sativa_Os01g0760600- 3 4 1 truncated A.thaliana_AT5G19550 5 6 1 A. thaliana_AT5G11520 7 8 1 A.thaliana_AT4G31990 9 10 1 A. thaliana_AT1G62800 11 12 1 B. napus_TA2320713 14 1 B. napus_TA23768 15 16 1 C. sinensis_TA12564 17 18 1 C.solstitialis_TA659 19 20 1 G. hirsutum_TA23799 21 22 1 G. max_AF03421023 24 1 G. raimondii_TA9413 25 26 1 H. annuus_TA8926 27 28 1 H.paradoxus_TA2606 29 30 1 J. regia_TA762 31 32 1 L. japonicus_TA1537 3334 1 L. perennis_TA512 35 36 1 L. perennis_TA605 37 38 1 N.tabacum_TA13125 39 40 1 P. glauca_TA15326 41 42 1 P. patens_136815 43 441 P. persica_TA3273 45 46 1 P. sitchensis_TA22265 47 48 1 P.trichocarpa_819551 49 50 1 P. trifoliata_TA8305 51 52 1 S.lycopersicum_TA38054 53 54 1 S. officinarum_TA26595 55 56 1 T.aestivum_TA52678 57 58 1 V. carteri_82929 59 60 1 V.vinifera_GSVIVT00016723001 61 62 1 V. vinifera_GSVIVT00032463001 63 64 1Z. mays_TA9042 65 66 1 C. reinhardtii_186959 67 68 2 C.solstitialis_TA2275 69 70 2 C. tinctorius_TA12 71 72 2 G.hirsutum_TA24406 73 74 2 G. max_TA61768 75 76 2 G. raimondii_TA9928 7778 2 H. exilis_TA1663 79 80 2 H. vulgare_BPS_7992 81 82 2 L.japonicus_TA1466 83 84 2 M. polymorpha_TA825 85 86 2 N. tabacum_TA1301587 88 2 O. sativa_Os02g0797500 89 90 2 P. glauca_TA14780 91 92 2 P.patens_102134 93 94 2 P. sitchensis_TA20968 95 96 2 P. taeda_TA6616 9798 2 P. trichocarpa_654206 99 100 2 P. trichocarpa_835828 101 102 2 P.vulgaris_TA4043 103 104 2 S. tuberosum_TA23192 105 106 2 V.carteri_81153 107 108 2 V. vinifera_GSVIVT00032723001 109 110 2 Z.mays_TA10886 111 112 2 A. thaliana_AT2G30970 113 114 4 C.sinensis_TA15250 115 116 4 G. max_TA50178 117 118 4 G. raimondii_TA9985119 120 4 H. vulgare_TA32835 121 122 4 H. vulgare_TA36301 123 124 4 O.lucimarinus_31597 125 126 4 O. sativa_Os02g0236000 127 128 4 O.sativa_Os06g0548000 129 130 4 O. taurii_32764 131 132 4 P. patens_169868133 134 4 P. sitchensis_TA23007 135 136 4 P. taeda_TA7145 137 138 4 V.vinifera_GSVIVT00018772001 139 140 4 V. vinifera_GSVIVT00037462001 141142 4 A. anophagefferens_21970 143 144 3 A. thaliana_AT2G22250.2 145 1463 B. napus_BPS_9867 147 148 3 C. reinhardtii_118364 149 150 3 G.hirsutum_TA27281 151 152 3 G. max_BPS_36342 153 154 3 H. vulgare_TA28738155 156 3 M. domestica_TA26867 157 158 3 N. tabacum_TA15308 159 160 3 O.basilicum_TA1043 161 162 3 O. sativa_Os01g0871300 163 164 3 P.patens_127152 165 166 3 P. pinaster_TA3616_71647 167 168 3 P.trichocarpa_scaff_V.183 169 170 3 P. trichocarpa_scaff_VII.574 171 172 3S. lycopersicum_TA37592 173 174 3 S. tuberosum_TA27739 175 176 3 T.aestivum_TA71539 177 178 3 V. carteri_103084 179 180 3 V.vinifera_GSVIVT00019453001 181 182 3 Z. mays_BPS_26636 183 184 3 Z.mays_BPS_4233 185 186 3 A. anophagefferens_21841 187 188 5 A.anophagefferens_27031 189 190 5 A. anophagefferens_27395 191 192 5 A.anophagefferens_58638 193 194 5 E. huxleyi_413787 195 196 5 E.huxleyi_437487 197 198 5 E. huxleyi_467854 199 200 5 P.tricornutum_23059 201 202 5 P. tricornutum_23871 203 204 5 T.pseudonana_269248 205 206 5

Alignment of polypeptide sequences was performed using the ClustalW 2.0algorithm of progressive alignment (Thompson et al. (1997) Nucleic AcidsRes 25:4876-4882; Chema et al. (2003). Nucleic Acids Res 31:3497-3500)with standard setting (slow alignment, similarity matrix: Gonnet, gapopening penalty 10, gap extension penalty: 0.2). Minor manual editingwas done to further optimise the alignment.

2.2. MYB91 Like Transcription Factor (MYB91)

Mutliple sequence alignment of all the MYB91 polypeptide sequences inTable A2 was performed using the ClsutalW 1.81 algorithm. Results of thealignment are shown in FIG. 5 of the present application. Two MYB DNAbinding domains with an InterPro accession number IPR014778, a MYBtranscription factor with an InterPro accession number IPR015495, and aC-terminal Conserved Domain, are marked with X's below the consensussequence.

2.3. Gibberellic Acid-Stimulated Arabidopsis (GASA)

Alignment of polypeptide sequences was performed using the AlignXprogramme from the Vector NTI (Invitrogen) which is based on the popularClustal W algorithm of progressive alignment (Thompson et al. (1997)Nucleic Acids Res 25:4876-4882; Chema et al. (2003). Nucleic Acids Res31:3497-3500). Default values are for the gap open penalty of 10, forthe gap extension penalty of 0.1 and the selected weight matrix isBlosum 62 (if polypeptides are aligned). Minor manual editing was doneto further optimise the alignment. Sequence conservation among GASApolypeptides is essentially in the C-terminal part of the polypeptides,the N-terminal part usually being more variable in sequence length andcomposition. The GASA polypeptides are aligned in FIG. 8.

2.4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

Alignment of polypeptide sequences was performed using the AlignXprogramme from the Vector NTI (Invitrogen), which is based on theClustalW 2.0 algorithm for progressive alignment (Thompson et al. (1997)Nucleic Acids Res 25:4876-4882; Chema et al. (2003). Nucleic Acids Res31:3497-3500); Alignment was performed with standard settings: gapopening penalty 10, gap extension penalty: 0.2. Minor manual editing wasdone to further optimise the alignment. The AUX/IAA polypeptides arealigned (FIG. 11).

Highly conserved amino acid residues are indicated in the consensussequence.

2.5. IAA14 Polypeptides

Alignment of polypeptide sequences was performed using the AlignXprogramme from the Vector NTI (Invitrogen) which is based on the popularClustal W algorithm of progressive alignment (Thompson et al. (1997)Nucleic Acids Res 25:4876-4882; Chema et al. (2003). Nucleic Acids Res31:3497-3500). Default values are for the gap open penalty of 10, forthe gap extension penalty of 0.1 and the selected weight matrix isBlosum 62 (if polypeptides are aligned). Minor manual editing was doneto further optimise the alignment. Sequence conservation amongIAA14-like polypeptides is essentially in the C-terminal half of thepolypeptides. The IAA14-like polypeptides are aligned in FIG. 14.

Example 3 Calculation of Global Percentage Identity Between PolypeptideSequences Useful in Performing the Methods of the Invention 3.1.Aspartate AminoTransferase (ASPAT)

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionare determined using one of the methods available in the art, the MatGAT(Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29.MatGAT: an application that generates similarity/identity matrices usingprotein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.Sequence similarity is shown in the bottom half of the dividing line andsequence identity is shown in the top half of the diagonal dividingline.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

A MATGAT table for local alignment of a specific domain, or data on %identity/similarity between specific domains may also be generated.

3.2. MYB91 Like Transcription Factor (MYB91)

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.Sequence similarity is shown in the bottom half of the dividing line andsequence identity is shown in the top half of the diagonal dividingline.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table C1 for the globalsimilarity and identity over the full length of the polypeptidesequences (excluding the partial polypeptide sequences).

The percentage identity between the full length polypeptide sequencesuseful in performing the methods of the invention can be as low as 52%amino acid identity compared to SEQ ID NO: 221.

TABLE C1 MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences of Table A. 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19 20 21 22 23 1. Antma_MYB91 72 64 64 63 68 69 7064 70 66 59 71 73 67 48 73 57 68 71 70 73 58 2. Aqufo_MYB91 84 70 68 6979 74 73 69 76 71 62 76 77 72 50 76 58 73 78 75 83 58 3. Arath_MYB91 7780 86 91 64 66 66 61 67 63 59 68 67 66 48 68 52 66 71 67 71 54 4.Brana_MYB91 76 80 92 85 63 67 64 62 66 63 59 67 66 65 47 67 52 65 70 6669 53 5. Carhi_MYB91 77 81 94 91 64 65 64 60 66 62 58 67 66 65 47 67 5065 69 67 69 52 6. Escca_MYB91 80 86 79 77 78 70 71 68 72 69 60 72 76 7150 73 57 71 73 72 79 56 7. Eucgr_MYB91 82 87 80 80 79 83 73 68 72 71 6474 77 71 50 75 54 72 76 74 79 57 8. Glyma_MYB91(a) 80 84 79 78 78 81 8477 73 76 67 73 76 89 49 74 55 88 76 72 80 57 9. Glyma_MYB91(b) 77 82 7677 75 78 82 84 69 73 71 67 70 77 51 68 52 76 72 67 74 55 10. Goshi_MYB9180 87 81 80 79 83 85 83 82 72 62 73 77 73 49 73 55 74 79 72 82 54 11.Lotco_MYB91(a) 77 83 77 77 77 80 84 84 84 83 69 70 73 76 51 72 55 75 7371 75 56 12. Lotco_MYB91(b) 72 75 72 71 71 72 78 78 80 75 80 62 65 68 4664 50 68 65 62 66 51 13. Lyces_MYB91 82 87 81 80 81 86 86 83 80 83 81 7576 72 49 92 56 73 75 98 80 55 14. Maldo_MYB91 84 87 79 81 78 84 88 85 8386 84 79 86 74 50 77 58 74 79 76 84 58 15. Medtr_MYB91 78 84 79 78 78 8283 93 85 83 86 79 83 84 49 72 55 96 76 71 78 57 16. Moral_MYB91 63 64 6465 64 63 65 63 63 62 63 59 63 62 62 49 46 49 52 49 50 44 17. Nicta_MYB9183 87 80 80 79 84 86 84 81 83 80 76 95 86 82 63 55 73 76 92 81 56 18.Orysa_MYB91 75 75 71 71 69 74 74 70 71 74 73 71 73 75 71 62 72 56 54 5557 62 19. Pissa_MYB91 79 85 80 79 78 82 83 92 85 83 85 79 83 84 98 63 8372 76 72 79 57 20. Poptr_MYB91 81 87 83 82 81 84 87 86 82 87 83 75 87 8785 65 87 73 85 74 86 56 21. Soltu_MYB91 83 86 80 80 80 85 86 82 80 82 8375 98 86 82 64 95 72 82 86 80 55 22. Vitvi_MYB91 84 91 83 82 82 88 89 8884 90 85 77 88 90 87 64 89 75 87 93 88 59 23. Zeama_MYB91 73 71 71 70 6970 72 73 70 70 71 65 71 71 71 64 72 73 71 73 70 71

The percentage amino acid identity can be significantly increased if themost conserved region of the polypeptides are compared. For example,when comparing the amino acid sequence of a MYB DNA transcription factorwith an InterPro entry IPR015495 as represented by SEQ ID NO: 268, or ofa MYB DNA binding domain with an InterPro accession number IPR014778 asrepresented by SEQ ID NO: 269 and/or 270, or of a C-terminal conserveddomain as represented by SEQ ID NO: 271 with the respectivecorresponding domains of the polypeptides of Table A1, the percentageamino acid identity increases significantly (in order of preference atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or moreamino acid sequence identity).

3.3. Gibberellic Acid-Stimulated Arabidopsis (GASA)

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.Sequence similarity is shown in the bottom half of the dividing line andsequence identity is shown in the top half of the diagonal dividingline.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table C2 for the globalsimilarity and identity over the full length of the polypeptidesequences. Percentage identity is given above the diagonal andpercentage similarity is given below the diagonal.

The percentage identity between the GASA polypeptide sequences useful inperforming the methods of the invention can be as low as 22.2% aminoacid identity compared to SEQ ID NO: 276.

TABLE C2 MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences. 1 2 3 4 5 6 7 8 9 10 11 12 13 1.TA5035_4679 42.0 35.5 27.6 35.0 29.9 35.9 52.1 33.0 28.2 35.9 64.3 36.62. TA5923_4679 52.1 48.0 34.2 35.6 32.0 33.3 47.1 31.1 28.1 33.3 40.836.1 3. Os05g0376800 40.8 55.9 28.8 26.7 28.4 27.6 38.6 23.2 26.6 27.634.2 23.9 4. Os04g0465300 37.1 47.1 40.1 24.2 33.1 37.4 28.8 30.6 35.137.4 29.5 33.0 5. Os10g0115550 42.7 49.6 42.1 35.0 30.7 33.3 34.6 23.729.4 32.5 36.8 30.5 6. AK105729 34.2 42.0 38.8 45.3 49.6 34.2 32.8 42.033.1 34.2 29.1 79.5 7. Os05g0432200 44.6 44.5 34.9 47.6 47.0 48.7 33.337.9 34.0 98.9 38.0 42.1 8. Os09g0414900 57.3 57.1 48.7 43.6 55.6 41.941.0 29.2 31.1 33.3 47.0 35.0 9. Os03g0607200 41.5 42.0 30.9 38.1 37.652.1 52.1 40.2 37.7 37.9 31.9 52.6 10. Os07g0592000 38.2 37.8 33.6 47.640.2 42.7 43.1 41.0 53.9 34.0 31.1 40.0 11. AK110640 43.5 44.5 34.9 47.646.2 48.7 98.9 41.0 52.1 43.1 38.0 42.1 12. Os06g0266800 73.8 46.2 38.236.2 44.4 30.8 44.6 53.0 40.4 36.3 43.5 35.4 13. Os03g0760800 43.0 46.231.6 46.7 44.4 79.5 59.1 41.0 64.9 51.0 59.1 40.9 14. scaff_205.30 41.243.7 38.2 49.5 39.3 47.9 46.1 47.9 52.9 53.9 46.1 37.3 55.9 15.scaff_II.204 35.6 45.4 37.5 53.3 42.7 49.6 60.4 42.7 48.5 46.1 59.4 37.656.4 16. scaff_II.2330 46.3 52.1 45.4 43.0 52.1 43.0 38.0 48.8 33.9 35.538.0 43.0 38.0 17. scaff_VI.397 60.0 62.2 49.3 48.6 49.6 45.3 43.0 54.743.0 41.2 42.0 59.0 48.0 18. scf_XVII.377 63.6 55.5 48.0 45.8 50.4 40.244.9 64.1 41.1 46.7 43.9 55.1 43.9 19. scaff_II.202 38.9 47.1 32.2 56.242.7 51.3 64.2 37.6 49.5 44.1 63.2 37.9 61.1 20. scaff_I.2410 44.8 41.230.3 40.0 42.7 47.9 53.3 38.5 53.2 48.0 52.2 47.1 57.0 21. scaff_I.148354.9 68.1 55.3 45.1 54.7 41.0 43.4 59.8 39.8 36.3 42.5 54.0 45.1 22.scaff_I.1926 18.4 26.1 30.6 26.1 22.4 22.4 22.0 23.3 21.2 20.0 21.6 18.019.2 23. scaff_XII.704 43.6 27.7 22.4 41.9 30.8 38.5 47.8 23.9 39.4 33.346.7 36.9 48.4 24. scaff_41.75 49.5 41.2 30.9 48.6 44.4 50.4 73.9 40.251.1 45.1 72.8 44.0 62.4 25. scaff_40.379 48.9 43.7 32.2 43.8 45.3 53.056.5 44.4 64.9 57.8 55.4 45.5 67.7 26. scaff_XV.507 39.8 39.5 28.3 48.637.6 41.9 55.9 36.8 42.6 44.1 54.8 38.7 52.7 27. scaff_II.203 43.6 29.424.3 36.2 32.5 38.5 54.3 28.2 40.4 34.3 53.3 41.7 47.3 28. scaff_II.232858.9 56.3 43.4 45.7 53.8 47.0 55.8 53.0 45.3 44.1 55.8 56.8 54.7 29.scaff_XIX.758 44.8 39.5 30.9 42.9 41.9 41.0 53.3 38.5 47.9 39.2 52.243.7 44.1 30. TA45751_4081 47.4 32.8 23.7 32.4 33.3 41.0 44.6 34.2 46.844.1 44.6 45.2 51.6 31. TA48119_4081 25.3 37.7 39.5 41.8 39.0 39.7 37.037.7 33.6 32.2 36.3 24.7 37.7 32. TA35962_4081 37.5 47.1 36.2 49.5 44.447.0 61.5 42.7 48.1 43.3 60.6 38.5 52.9 33. BI208422 65.4 50.4 40.8 40.046.2 36.8 43.5 48.7 40.4 43.1 43.5 63.1 46.2 34. BG128975 51.8 64.7 50.050.0 58.1 44.4 44.6 62.4 40.2 35.7 43.8 50.9 43.8 35. TA52374_4081 36.646.2 35.5 53.6 47.0 47.9 58.0 46.2 46.4 44.6 57.1 39.3 52.7 36.TA37180_4081 57.3 55.5 45.4 45.7 50.4 43.6 49.0 53.0 42.7 49.0 49.0 56.350.0 37. BE353147 39.2 44.5 37.5 59.0 36.8 49.6 59.8 40.2 47.1 41.2 58.837.3 52.9 38. TA56938_4081 62.5 60.5 46.7 49.5 47.9 41.9 48.1 60.7 42.349.0 47.1 55.8 50.0 39. BG130916 70.5 48.7 38.2 40.0 39.3 36.8 40.2 46.236.2 37.3 39.1 59.5 45.2 40. SEQ ID NO: 276 51.8 68.1 50.7 48.2 52.144.4 45.6 58.1 44.7 42.1 44.7 50.0 45.6 41. TA41886_4081 37.9 45.4 34.959.0 35.9 52.1 58.3 38.5 45.6 39.8 58.3 37.9 56.3 42. TA36295_4081 46.645.4 35.5 49.5 47.0 41.0 53.4 41.9 47.6 45.6 52.4 41.7 55.3 43.TA56201_4081 50.0 44.5 36.2 47.6 41.9 41.0 43.6 47.0 44.7 45.1 43.6 43.651.1 44. AJ785329 52.6 31.9 24.3 26.7 29.1 23.9 30.4 34.2 26.6 28.4 30.447.6 30.1 45. CA725087 49.1 54.6 41.4 37.9 49.6 36.8 41.4 56.4 35.3 42.241.4 55.2 39.7 46. TA69823_4565 24.4 30.3 29.9 25.4 29.4 33.3 25.4 27.928.4 38.8 25.4 20.4 28.4 47. TA53297_4565 43.5 42.9 32.9 50.5 46.2 47.087.0 37.6 48.9 36.3 85.9 46.7 58.1 48. TA101332_4565 50.5 55.5 40.1 47.663.2 47.9 55.3 56.4 45.6 47.6 54.4 48.5 50.5 49. TA66036_4565 44.7 44.534.9 47.6 39.3 73.5 59.6 40.2 63.8 53.9 59.6 42.6 90.4 50. TA100367_456555.3 49.6 45.4 44.7 47.9 37.6 40.4 59.0 36.8 42.1 39.5 62.3 38.6 51.TA92393_4565 60.4 55.5 42.1 43.8 51.3 41.0 48.5 58.1 42.6 48.0 48.5 73.344.6 52. BM136027 43.6 45.4 34.2 47.6 40.2 72.6 58.5 40.2 62.8 55.9 58.542.6 89.4 53. CA705831 33.6 42.0 32.2 38.1 47.9 65.0 44.2 41.0 48.7 43.444.2 35.4 69.0 54. CA593033 29.7 38.3 31.6 35.2 41.4 60.2 40.6 36.7 44.540.6 40.6 28.1 61.7 55. CK153563 60.6 53.8 40.8 41.9 49.6 38.5 52.1 57.344.7 45.1 52.1 68.1 47.9 56. TA66038_4565 40.8 45.4 33.6 42.9 38.5 70.958.2 41.0 63.3 52.0 58.2 41.8 85.7 57. TA52915_4565 43.5 41.2 32.2 51.445.3 46.2 85.9 38.5 47.9 36.3 84.8 46.7 58.1 58. TA69821_4565 41.1 41.238.2 40.2 47.0 46.2 46.7 44.4 49.5 75.7 46.7 34.6 50.5 59. TA95153_456530.8 38.7 34.9 41.0 39.3 40.2 47.0 36.8 39.3 36.8 46.2 35.9 41.9 60.CD899399 39.8 44.5 32.9 42.9 38.5 73.5 57.1 40.2 62.2 52.9 57.1 39.888.8 61. TA77646_4565 61.6 57.1 43.4 44.8 51.3 38.5 50.5 59.0 41.4 50.050.5 70.7 48.5 62. TA51752_4565 29.5 39.5 37.5 34.9 34.9 42.6 44.2 38.838.8 35.7 43.4 31.0 38.0 63. Pop_GASA 49.4 43.7 32.2 42.9 43.6 48.7 53.342.7 58.5 52.0 52.2 47.2 60.2 64. Mt_GASA 36.6 43.7 36.8 50.9 47.9 45.350.0 42.7 50.0 44.6 49.1 36.6 48.2 65. At2g30810 57.5 61.3 45.4 50.955.6 45.3 43.4 60.7 39.6 45.3 42.5 51.9 46.2 66. At3g02885 62.9 58.046.1 46.7 54.7 44.4 54.6 57.3 47.4 48.0 53.6 59.8 50.5 67. At5g1523057.5 53.8 40.8 43.4 52.1 42.7 44.3 55.6 42.5 43.4 43.4 54.7 43.4 68.At1g74670 62.4 60.5 45.4 45.7 49.6 40.2 52.5 57.3 41.6 49.0 51.5 58.444.6 14 15 16 17 18 19 20 21 22 23 24 25 26 1. TA5035_4679 31.1 29.738.8 51.0 56.1 31.6 34.5 48.7 12.2 36.7 37.6 37.5 32.3 2. TA5923_467934.2 36.1 43.8 55.5 46.2 37.0 28.6 54.6 17.6 22.5 29.4 31.1 27.5 3.Os05g0376800 28.3 28.3 36.8 42.8 38.2 26.3 22.4 49.3 22.0 18.3 23.0 24.321.6 4. Os04g0465300 34.3 39.0 32.8 32.4 33.6 42.9 28.6 31.6 21.4 36.234.0 32.4 39.0 5. Os10g0115550 27.4 28.6 35.6 39.0 38.3 31.9 32.5 34.914.0 27.1 32.5 34.2 32.2 6. AK105729 39.3 36.0 31.0 32.5 32.5 41.5 40.230.6 16.3 30.5 41.0 47.0 33.9 7. Os05g0432200 34.3 49.0 28.9 34.0 34.653.1 38.0 32.7 18.0 43.0 59.8 41.3 48.4 8. Os09g0414900 32.5 29.9 38.845.3 52.1 29.1 29.1 48.7 16.3 20.3 30.8 33.3 25.4 9. Os03g0607200 37.135.6 25.8 27.1 28.2 37.8 38.3 29.3 14.5 31.6 35.1 47.9 33.3 10.Os07g0592000 39.3 33.0 29.3 30.8 33.6 35.0 35.0 27.0 14.6 24.5 31.1 42.731.7 11. AK110640 34.3 48.0 28.9 34.0 34.6 52.1 38.0 32.7 17.6 41.9 58.741.3 47.3 12. Os06g0266800 32.4 29.8 38.8 51.0 49.5 32.6 35.6 50.4 13.532.9 37.4 39.8 34.4 13. Os03g0760800 44.9 43.3 30.6 35.9 34.2 44.9 47.333.6 13.7 38.3 51.1 60.6 39.6 14. scaff_205.30 35.9 28.9 33.0 36.1 37.349.0 33.6 16.7 26.2 32.4 54.9 31.1 15. scaff_II.204 44.1 29.8 31.7 31.877.2 31.7 37.4 19.6 39.2 43.6 34.7 49.0 16. scaff_II.2330 38.0 37.2 39.740.5 28.9 24.8 41.3 17.1 23.8 30.6 30.6 25.4 17. scaff_VI.397 46.1 47.547.1 49.5 35.0 32.0 61.4 18.4 27.7 36.3 37.0 26.7 18. scf_XVII.377 53.348.6 45.5 60.7 33.6 29.9 54.0 13.5 25.0 34.6 38.9 33.3 19. scaff_II.20245.1 85.1 32.2 47.0 43.0 33.3 33.6 18.0 46.9 47.4 36.1 53.1 20.scaff_I.2410 55.9 43.6 40.5 44.0 45.8 46.3 31.0 14.3 30.7 36.3 62.5 35.721. scaff_I.1483 46.0 48.7 52.1 64.6 62.8 44.2 45.1 15.5 24.6 33.6 36.330.7 22. scaff_I.1926 22.4 24.9 22.4 23.3 21.2 21.2 20.4 24.5 19.2 15.915.5 21.5 23. scaff_XII.704 31.4 44.6 26.4 34.0 29.9 51.6 40.2 30.1 19.641.3 32.6 63.8 24. scaff_41.75 47.1 58.4 39.7 48.0 47.7 61.1 50.5 47.821.2 48.4 41.8 45.2 25. scaff_40.379 58.8 44.6 39.7 47.0 50.5 47.4 72.745.1 19.6 39.8 50.5 36.2 26. scaff_XV.507 37.3 61.4 38.8 40.0 43.0 67.449.5 40.7 23.7 67.7 57.0 46.2 27. scaff_II.203 33.3 55.4 30.6 37.0 30.856.8 39.1 34.5 18.0 67.6 57.1 40.9 52.7 28. scaff_II.2328 49.0 49.5 60.362.0 57.9 46.3 50.5 61.9 22.0 34.7 53.7 53.7 47.4 29. scaff_XIX.758 37.351.5 40.5 46.0 43.9 56.8 43.7 40.7 23.7 52.9 58.2 50.0 55.9 30.TA45751_4081 51.0 32.7 30.6 40.0 37.4 35.8 63.2 31.9 14.7 51.5 42.9 67.036.6 31. TA48119_4081 35.6 45.9 37.0 32.2 31.5 44.5 33.6 37.0 32.2 41.837.7 31.5 52.7 32. TA35962_4081 40.4 75.0 37.2 46.2 44.9 75.0 41.3 50.424.9 47.1 61.5 43.3 58.7 33. BI208422 45.1 42.6 50.4 58.0 54.2 43.2 46.056.6 18.8 37.0 48.4 51.1 44.1 34. BG128975 45.5 49.1 52.9 69.6 59.8 42.942.9 78.8 24.9 29.5 49.1 48.2 39.3 35. TA52374_4081 49.1 62.5 38.0 44.652.7 59.8 39.3 52.2 25.7 42.0 54.5 42.9 52.7 36. TA37180_4081 48.0 46.555.4 63.0 61.7 49.0 47.9 62.8 22.4 31.3 49.0 50.0 46.9 37. BE353147 48.061.8 34.7 48.0 43.0 63.7 41.2 47.8 24.1 45.1 56.9 46.1 55.9 38.TA56938_4081 54.8 52.9 51.2 62.5 84.1 47.1 47.1 66.4 24.9 32.7 48.1 50.043.3 39. BG130916 38.2 38.6 46.3 60.0 47.7 38.9 47.1 54.0 18.4 44.4 45.144.3 38.7 40. SEQ ID NO: 276 47.4 45.6 52.1 71.1 60.5 43.9 43.9 71.926.9 27.2 42.1 44.7 38.6 41. TA41886_4081 51.5 61.2 38.8 44.7 41.1 64.139.8 46.9 26.1 45.6 58.3 43.7 59.2 42. TA36295_4081 42.7 58.3 39.7 44.748.6 54.4 45.6 48.7 24.5 42.7 57.3 49.5 55.3 43. TA56201_4081 50.0 45.538.8 48.0 51.4 43.2 53.2 49.6 18.8 29.8 46.8 51.1 38.3 44. AJ785329 29.426.7 29.8 36.0 35.5 29.5 35.6 34.5 12.2 43.9 31.9 35.2 33.3 45. CA72508742.2 39.7 49.6 47.4 57.8 38.8 38.8 60.3 19.2 23.3 42.2 38.8 35.3 46.TA69823_4565 27.9 26.9 27.4 25.9 26.9 23.4 25.9 28.9 26.5 15.9 24.4 28.924.4 47. TA53297_4565 41.2 60.4 41.3 42.0 47.7 66.3 47.8 47.8 22.0 47.872.8 48.9 58.1 48. TA101332_4565 46.6 49.5 45.5 57.3 57.0 55.3 46.6 56.624.5 35.0 52.4 52.4 47.6 49. TA66036_4565 55.9 52.5 39.7 49.0 43.9 56.859.6 46.0 20.4 45.7 58.5 61.7 51.1 50. TA100367_4565 43.9 47.4 47.1 55.360.5 43.9 40.4 62.3 22.4 26.3 43.0 42.1 36.0 51. TA92393_4565 47.1 48.551.2 59.4 67.3 46.5 47.5 64.6 19.6 29.7 50.5 45.5 40.6 52. BM136027 54.955.4 38.8 49.0 44.9 57.9 59.6 46.0 20.4 45.7 57.4 60.6 51.1 53. CA70583150.4 46.0 38.8 38.9 41.6 43.4 45.1 44.2 18.0 31.0 44.2 50.4 38.9 54.CA593033 45.3 39.8 33.6 32.8 33.6 38.3 40.6 37.5 17.1 29.7 39.8 45.336.7 55. 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TA36295_4081 48.2 55.3 33.726.0 31.7 16.3 43.0 40.0 37.4 36.0 37.9 36.4 43. TA56201_4081 48.2 42.749.5 45.7 33.3 22.1 34.0 45.7 38.4 34.2 37.3 38.8 44. AJ785329 35.1 25.229.1 50.0 27.4 12.4 24.7 33.7 26.3 28.7 35.3 26.3 45. CA725087 53.4 43.142.2 44.0 31.0 16.1 29.7 39.2 28.0 54.2 73.7 28.8 46. TA69823_4565 30.324.9 25.4 26.4 15.9 22.4 18.8 21.8 21.6 19.3 17.8 21.1 47. TA53297_456543.9 61.2 56.3 44.7 27.2 39.7 21.4 40.8 44.2 29.8 39.8 43.2 48.TA101332_4565 56.1 48.5 49.5 55.3 37.9 48.3 28.4 54.4 39.6 38.6 48.539.6 49. TA66036_4565 43.9 52.4 52.4 48.9 28.7 33.6 26.4 57.4 47.6 34.234.6 98.9 50. TA100367_4565 57.9 45.6 45.6 46.5 35.1 67.2 25.4 41.2 49.143.0 68.4 35.0 51. TA92393_4565 60.5 49.5 48.5 49.5 39.6 76.7 23.4 49.558.3 39.6 74.6 35.6 52. BM136027 43.9 51.5 51.5 53.2 28.7 34.5 25.9 56.447.6 98.9 42.1 40.6 53. CA705831 43.0 44.2 41.6 36.3 20.4 42.2 27.4 43.443.4 69.0 43.9 40.7 68.1 54. CA593033 37.5 39.8 37.5 33.6 21.1 37.5 28.438.3 38.3 61.7 39.1 32.8 60.9 55. CK153563 56.1 49.5 46.6 56.4 41.5 70.724.4 53.2 56.3 42.6 63.2 85.1 42.6 56. TA66038_4565 46.5 48.5 48.5 49.028.6 35.3 28.9 51.0 51.5 82.7 44.7 47.5 81.6 57. TA52915_4565 44.7 60.257.3 45.7 27.2 39.7 21.4 98.9 54.4 56.4 41.2 49.5 55.3 58. TA69821_456545.6 39.3 44.9 48.6 29.0 38.8 48.3 39.3 58.9 52.3 44.7 43.9 51.4 59.TA95153_4565 44.4 44.4 43.6 37.6 23.1 35.0 25.4 47.9 41.9 41.0 35.9 38.540.2 60. CD899399 45.6 48.5 49.5 49.0 27.6 37.1 27.4 52.0 51.5 87.8 43.944.6 86.7 61. TA77646_4565 57.0 51.5 50.5 52.5 40.4 80.2 24.4 51.5 59.241.4 71.9 94.1 42.4 62. TA51752_4565 41.9 40.3 40.3 38.0 20.2 31.8 28.944.2 41.1 38.8 34.9 34.9 36.4 63. Pop_GASA 45.6 44.7 48.5 53.2 36.0 42.227.9 43.5 48.5 58.5 43.0 50.5 58.5 64. Mt_GASA 48.2 49.1 54.5 41.1 28.643.1 26.4 49.1 47.3 46.4 41.2 44.6 46.4 65. At2g30810 61.4 48.1 49.155.7 35.8 50.0 27.9 47.2 66.0 44.3 57.9 60.4 47.2 66. At3g02885 55.346.6 48.5 52.6 36.1 54.3 24.9 51.5 66.0 51.5 55.3 65.3 52.6 67.At5g15230 56.1 43.4 52.8 51.9 37.7 55.2 26.9 43.4 57.5 43.4 56.1 64.244.3 68. At1g74670 63.2 43.7 53.4 51.5 37.6 56.0 24.4 47.5 57.3 48.554.4 64.4 48.5 53 54 55 56 57 58 59 60 61 62 63 64 65 1. TA5035_467930.1 25.8 51.1 36.7 34.8 30.6 23.9 35.7 52.5 21.7 34.8 26.8 48.6 2.TA5923_4679 29.1 26.3 45.4 35.2 30.3 30.6 29.3 34.4 47.1 29.5 32.8 32.547.9 3. Os05g0376800 20.7 19.6 32.2 27.1 26.3 28.6 24.0 25.8 33.6 27.923.7 27.5 35.5 4. Os04g0465300 27.6 24.6 33.3 31.5 40.0 31.2 32.5 33.333.3 27.9 32.4 38.1 36.7 5. Os10g0115550 32.5 27.7 43.6 29.9 32.5 32.225.0 29.1 46.2 22.0 33.1 31.3 43.9 6. AK105729 51.5 47.0 30.8 64.4 36.836.1 30.8 68.6 30.6 33.6 43.6 31.4 31.6 7. Os05g0432200 30.7 27.9 38.344.9 70.7 33.3 37.3 42.9 38.4 34.9 36.6 38.6 34.9 8. Os09g0414900 25.923.4 46.2 32.5 29.1 32.8 24.4 31.7 51.3 29.2 30.8 28.9 50.0 9.Os03g0607200 40.4 37.2 31.7 51.5 34.7 36.6 31.1 51.5 32.4 29.0 40.4 33.027.5 10. Os07g0592000 32.3 31.2 32.0 40.2 31.1 70.0 27.7 41.1 34.3 26.939.8 28.4 32.4 11. AK110640 30.7 27.9 38.3 44.9 69.6 33.3 36.4 42.9 38.434.1 36.6 37.7 34.9 12. Os06g0266800 31.0 24.4 64.9 36.7 37.6 29.6 29.134.7 67.7 24.0 37.1 28.6 46.2 13. Os03g0760800 63.7 57.0 39.2 78.6 45.341.4 32.8 82.7 37.5 29.8 54.8 34.8 34.9 14. scaff_205.30 39.4 36.6 37.942.9 30.4 39.8 29.1 44.6 40.8 29.0 55.3 31.9 33.9 15. scaff_II.204 34.129.7 37.6 37.5 50.0 32.4 32.5 39.4 37.9 29.5 33.7 43.4 37.0 16.scaff_II.2330 27.3 22.8 41.5 31.5 29.8 30.9 25.2 31.5 41.5 22.5 29.834.7 43.0 17. scaff_VI.397 25.4 22.6 47.0 34.0 32.0 32.4 27.4 33.0 48.026.4 32.0 31.3 46.2 18. scf_XVII.377 30.2 24.3 49.5 35.5 36.4 35.5 24.834.5 53.3 25.6 32.7 31.6 49.5 19. scaff_II.202 34.2 28.8 39.6 38.8 54.733.3 33.3 40.8 39.4 32.6 34.7 40.2 36.1 20. scaff_I.2410 38.1 35.2 40.050.0 36.6 32.4 29.7 50.0 36.4 26.9 69.2 31.9 31.2 21. scaff_I.1483 28.125.3 51.3 35.3 33.6 32.2 28.6 35.3 54.0 30.2 34.5 35.3 49.6 22.scaff_I.1926 11.6 8.6 15.9 14.5 19.2 17.4 16.7 15.3 16.3 17.5 15.9 22.417.1 23. scaff_XII.704 24.6 22.5 28.4 33.3 44.1 22.9 25.4 33.3 27.0 23.830.0 42.9 25.2 24. scaff_41.75 35.4 30.5 43.2 46.9 56.5 34.5 32.5 48.042.6 30.2 36.3 37.5 36.8 25. scaff_40.379 44.2 40.6 43.6 57.6 39.1 40.727.4 54.1 40.4 25.6 71.9 36.6 34.0 26. scaff_XV.507 28.2 25.8 34.7 36.448.4 27.5 32.2 38.2 36.0 30.0 30.1 56.3 30.8 27. scaff_II.203 27.4 22.734.0 34.7 50.0 25.0 25.6 35.7 30.3 22.5 31.5 34.8 30.2 28. scaff_II.232833.3 27.3 56.7 38.8 38.9 35.2 29.2 38.8 55.9 27.9 38.9 35.7 52.8 29.scaff_XIX.758 25.7 21.1 37.2 32.7 42.4 24.8 27.4 33.7 34.3 24.0 36.042.9 33.0 30. TA45751_4081 40.7 37.5 37.2 49.0 32.6 33.3 23.9 49.0 35.422.5 57.3 26.8 29.2 31. TA48119_4081 22.0 20.2 23.3 27.5 32.2 26.4 29.927.5 25.3 26.0 22.6 43.0 27.4 32. TA35962_4081 29.4 26.2 33.3 35.5 48.130.6 37.6 37.4 36.5 34.1 34.6 40.2 36.1 33. BI208422 30.1 23.4 51.1 34.733.7 28.7 24.8 34.7 49.5 21.7 36.0 35.7 47.2 34. BG128975 28.9 25.3 50.030.4 36.6 33.3 25.6 30.4 50.0 25.6 27.7 33.6 49.6 35. TA52374_4081 32.830.2 37.5 38.3 43.8 32.5 34.2 40.9 43.8 33.3 35.7 40.7 39.1 36.TA37180_4081 31.4 24.8 51.0 34.3 37.5 30.6 25.6 33.3 49.5 24.0 35.4 36.649.1 37. BE353147 28.2 24.5 34.3 33.3 49.0 32.1 35.9 35.2 34.0 31.0 32.436.6 38.9 38. TA56938_4081 33.1 26.8 54.8 35.5 36.5 36.1 28.2 36.4 57.726.4 36.8 33.0 52.8 39. BG130916 28.3 24.2 47.9 33.7 33.7 27.8 23.1 32.744.0 23.3 36.0 27.7 43.4 40. SEQ ID NO: 276 27.2 24.5 46.5 35.9 34.234.5 32.5 35.0 48.2 31.0 32.5 34.8 50.9 41. TA41886_4081 28.8 25.0 41.034.0 47.6 33.3 31.6 34.9 39.8 29.5 35.9 36.2 33.0 42. TA36295_4081 30.427.1 38.5 36.9 43.0 30.6 31.6 36.9 39.8 27.9 36.5 44.6 38.7 43.TA56201_4081 28.4 26.7 42.1 40.2 35.1 39.4 28.2 40.4 41.0 30.2 39.4 33.038.3 44. AJ785329 19.3 19.4 37.9 26.3 24.7 22.2 18.8 25.3 36.0 16.3 28.921.4 31.8 45. CA725087 32.2 29.2 68.1 30.5 29.7 25.8 23.3 30.5 78.4 21.431.4 27.1 39.0 46. TA69823_4565 19.3 19.6 17.8 21.4 18.3 46.8 18.7 20.619.3 22.3 19.8 19.2 21.8 47. TA53297_4565 30.7 27.1 41.7 42.9 97.8 30.834.2 44.9 40.6 33.3 31.5 36.8 34.9 48. TA101332_4565 32.8 28.6 47.6 36.840.8 40.2 30.8 37.7 50.5 32.6 36.9 33.6 49.5 49. TA66036_4565 65.5 58.637.1 79.6 43.2 40.2 31.1 83.7 35.3 26.7 53.2 35.7 34.9 50. TA100367_456530.9 27.8 57.9 35.3 30.7 33.6 27.4 34.5 65.8 27.1 32.5 27.8 49.6 51.TA92393_4565 33.3 26.8 84.2 38.7 39.8 33.3 30.8 37.5 94.1 26.4 38.8 33.950.9 52. BM136027 64.6 57.8 37.1 78.6 42.1 39.3 30.3 82.7 36.3 28.2 53.235.7 37.6 53. CA705831 81.3 35.3 65.8 31.6 31.0 25.4 68.4 33.9 24.0 45.126.9 31.3 54. CA593033 82.8 28.2 60.6 27.9 30.1 23.5 61.4 27.2 22.4 41.423.5 24.5 55. CK153563 40.7 32.8 41.6 37.2 32.4 28.2 41.6 87.9 26.4 40.431.3 50.0 56. TA66038_4565 71.7 65.6 50.0 42.9 39.3 27.7 94.9 38.2 28.252.0 32.2 34.9 57. TA52915_4565 43.4 38.3 52.1 51.0 30.8 34.2 44.9 40.632.6 31.5 36.3 35.8 58. TA69821_4565 44.2 40.6 42.1 49.5 39.3 28.8 38.233.3 27.7 37.6 27.8 34.2 59. TA95153_4565 41.0 38.3 35.9 38.5 48.7 39.328.6 29.9 76.7 31.6 31.4 26.5 60. CD899399 72.6 64.8 49.0 96.9 52.0 47.736.8 38.2 29.0 53.1 33.0 36.7 61. TA77646_4565 41.6 33.6 88.9 44.4 51.545.8 38.5 46.5 27.1 39.6 33.9 51.9 62. TA51752_4565 38.0 38.8 34.1 38.845.0 39.5 82.9 37.2 34.9 30.2 30.0 27.1 63. Pop_GASA 50.4 45.3 52.1 61.244.6 49.5 39.3 61.2 52.5 39.5 32.1 40.2 64. Mt_GASA 43.4 39.1 41.1 46.448.2 42.0 43.6 47.3 46.4 42.6 46.4 33.0 65. At2g30810 41.6 33.6 58.545.3 48.1 45.8 41.0 45.3 60.4 40.3 53.8 48.2 66. At3g02885 46.9 38.364.9 51.0 51.5 45.8 41.0 52.0 66.7 37.2 54.6 47.3 61.3 67. At5g1523038.1 33.6 62.3 48.1 42.5 43.0 39.3 48.1 65.1 38.8 46.2 49.1 55.7 68.At1g74670 42.5 37.5 64.4 48.5 46.5 45.8 40.2 47.5 68.3 39.5 50.5 47.364.2 66 67 68 1. TA5035_4679 48.0 50.0 59.4 2. TA5923_4679 47.9 45.049.6 3. Os05g0376800 35.5 32.2 39.5 4. Os04g0465300 37.6 33.6 34.6 5.Os10g0115550 42.7 37.5 40.7 6. AK105729 33.9 31.7 34.2 7. Os05g043220039.2 31.1 37.6 8. Os09g0414900 47.0 41.9 47.0 9. Os03g0607200 34.0 30.327.9 10. Os07g0592000 38.8 27.9 33.3 11. AK110640 39.2 31.1 37.6 12.Os06g0266800 52.6 49.1 48.5 13. Os03g0760800 41.0 30.3 35.6 14.scaff_205.30 35.3 33.0 35.9 15. scaff_II.204 35.6 32.7 38.8 16.scaff_II.2330 45.5 36.4 40.5 17. scaff_VI.397 54.0 44.4 54.5 18.scf_XVII.377 55.5 67.6 63.6 19. scaff_II.202 40.2 33.0 38.2 20.scaff_I.2410 35.1 31.1 31.7 21. scaff_I.1483 51.3 52.2 53.1 22.scaff_I.1926 15.9 14.7 14.7 23. scaff_XII.704 27.6 25.2 29.4 24.scaff_41.75 43.3 33.0 37.6 25. scaff_40.379 44.3 38.7 35.6 26.scaff_XV.507 37.0 30.8 38.2 27. scaff_II.203 32.0 25.5 30.7 28.scaff_II.2328 64.9 47.2 54.5 29. scaff_XIX.758 36.1 34.0 35.6 30.TA45751_4081 33.0 29.2 31.7 31. TA48119_4081 28.8 24.0 27.4 32.TA35962_4081 35.6 34.0 38.5 33. BI208422 57.7 45.3 54.5 34. BG12897554.0 47.8 53.6 35. TA52374_4081 33.3 31.3 36.8 36. TA37180_4081 61.049.5 58.3 37. BE353147 34.6 31.8 34.3 38. TA56938_4081 55.1 63.2 64.439. BG130916 48.5 42.5 48.5 40. SEQ ID NO: 276 46.5 43.9 53.5 41.TA41886_4081 35.9 33.3 34.0 42. TA36295_4081 35.9 40.0 41.3 43.TA56201_4081 42.4 37.6 40.2 44. AJ785329 32.7 32.7 33.3 45. CA72508745.3 42.7 42.4 46. TA69823_4565 21.8 17.8 21.3 47. TA53297_4565 41.235.8 32.7 48. TA101332_4565 51.9 41.5 45.6 49. TA66036_4565 41.0 30.335.6 50. TA100367_4565 43.6 45.6 42.1 51. TA92393_4565 53.8 54.2 49.552. BM136027 42.0 31.2 35.6 53. CA705831 36.1 23.4 31.7 54. CA59303329.9 21.0 27.5 55. CK153563 55.7 52.3 51.5 56. TA66038_4565 39.2 33.036.5 57. TA52915_4565 41.2 34.9 32.7 58. TA69821_4565 37.0 28.7 33.3 59.TA95153_4565 31.1 29.1 30.8 60. CD899399 41.7 33.0 35.6 61. TA77646_456558.6 54.2 53.5 62. TA51752_4565 28.7 28.7 26.4 63. Pop_GASA 38.4 33.038.2 64. Mt_GASA 33.0 30.1 33.9 65. At2g30810 50.9 45.3 50.9 66.At3g02885 50.0 54.4 67. At5g15230 61.3 57.5 68. At1g74670 65.3 67.9

3.4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.

Parameters that may be used in the comparison:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

3.5. IAA14 Polypeptides

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.Sequence similarity is shown in the bottom half of the dividing line andsequence identity is shown in the top half of the diagonal dividingline.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table C3 for the globalsimilarity and identity over the full length of the polypeptidesequences. Percentage identity is given above the diagonal andpercentage similarity is given below the diagonal.

The percentage identity between the IAA14-like polypeptide sequencesuseful in performing the methods of the invention can be as low as 26.3%amino acid identity compared to SEQ ID NO: 738 (A.thaliana_AT4G14550.1), but is usually above 35%.

TABLE C3 MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences. 1 2 3 4 5 6 7 8 9 10 11 12 1. A.thaliana_AT4G14550.1#1 80.7 68.6 63.6 70.5 73.7 67.2 66.8 26.3 54.5 61.464.2 2. A. thaliana_AT3G23050.1#1 84.8 86.4 63.3 68.1 70.2 63.9 63.524.7 55.8 60.2 64.3 3. A. thaliana_AT3G23050.2#1 76.3 86.4 53.6 57.558.6 53.5 53.6 12.0 45.0 49.2 53.2 4. P. trichocarpa_566151#1 72.2 72.961.4 85.6 66.9 63.3 57.3 21.3 56.6 57.8 54.5 5. P. trichocarpa_720961#179.0 81.0 68.5 87.4 74.3 70.9 63.9 23.8 54.2 63.6 59.9 6. M.truncatula_TA20354_3880#1 81.4 79.8 69.1 75.8 83.5 72.3 64.4 26.3 55.861.2 63.9 7. S. lycopersicum_TA40922_4081#1 76.3 77.4 66.9 70.4 78.283.1 60.5 24.6 55.3 59.4 62.5 8. A. thaliana_AT1G04250.1#1 82.5 77.067.7 67.5 75.4 77.1 74.6 22.3 50.2 55.5 59.3 9. O. sativa_CB657009#127.2 26.3 15.2 23.1 25.4 26.7 25.8 26.6 23.8 24.1 25.4 10. O.sativa_TA41733_4530#1 64.6 66.8 55.2 71.1 67.9 66.8 63.9 59.9 23.8 55.754.2 11. M. truncatula_TA20951_3880#1 71.9 73.1 60.5 69.0 76.3 71.5 69.668.4 25.7 67.1 63.5 12. A. thaliana_AT3G04730.1#1 75.8 77.8 66.5 67.975.8 78.8 77.1 74.6 27.5 64.6 73.5 13. S. lycopersicum_TA48108_4081#169.7 67.9 63.3 62.1 68.5 72.0 69.5 71.2 29.8 56.0 66.8 71.6 14. M.truncatula_TA27011_3880#1 58.5 59.5 51.2 61.5 60.9 59.2 57.5 54.8 18.755.2 58.5 58.2 15. M. truncatula_TA22814_3880#1 71.0 72.2 60.0 65.7 72.274.3 71.4 71.4 25.7 59.6 70.4 71.4 16. P. trichocarpa_643213#1 75.9 75.764.6 68.2 74.2 79.7 78.9 74.3 27.0 66.4 73.9 76.8 17. A.thaliana_AT3G23030.1#1 51.8 48.1 47.6 44.0 48.8 50.8 49.2 52.8 25.3 44.047.8 48.7 18. A. thaliana_AT4G14560.1#1 53.5 48.6 48.6 46.2 50.0 53.051.3 54.1 29.8 45.5 50.6 52.1 19. A. thaliana_AT1G04240.1#1 54.8 53.553.3 46.9 53.6 54.7 53.0 55.0 26.5 47.7 53.8 54.2 20. S.lycopersicum_TA38817_4081#1 54.8 52.7 52.4 46.2 51.2 49.2 51.3 52.0 23.744.8 50.2 50.4 21. S. lycopersicum_TA43058_4081#1 55.3 53.1 53.3 48.455.6 53.8 51.3 56.8 23.0 47.3 53.4 55.1 22. P. trichocarpa_726443#1 54.453.5 53.8 43.3 48.8 52.1 49.2 55.0 24.0 48.0 48.6 55.5 23. P.trichocarpa_564913#1 57.9 52.7 51.4 48.7 51.6 54.7 53.4 59.8 23.2 51.651.4 53.8 24. P. trichocarpa_831610#1 57.9 56.0 55.7 49.8 54.8 56.4 55.557.2 25.1 50.2 53.0 58.1 25. P. trichocarpa_798526#1 56.6 55.1 54.8 48.454.8 57.6 55.9 57.6 23.6 49.1 53.8 57.6 26. M. truncatula_TA20557_3880#155.7 53.9 53.8 44.8 50.4 52.5 51.7 55.0 26.4 47.3 50.6 53.0 27. M.truncatula_TA20558_3880#1 55.3 49.8 49.0 46.9 53.6 51.7 53.8 54.1 26.348.0 50.6 55.9 28. P. trichocarpa_823671#1 58.3 53.9 54.3 48.0 54.0 56.454.7 57.6 23.2 49.8 53.8 55.5 29. P. trichocarpa_595419#1 57.0 55.1 55.747.3 53.6 55.9 54.7 55.9 23.4 48.4 52.6 53.8 30. M.truncatula_TA31746_3880#1 56.6 55.1 54.8 49.5 54.0 53.8 53.8 58.5 25.048.4 54.2 55.9 31. S. lycopersicum_TA42190_4081#1 54.4 53.9 52.9 49.555.2 55.9 53.8 54.1 25.9 50.9 54.2 55.5 32. A. thaliana_AT4G29080.1#153.1 54.4 44.9 57.7 55.1 54.4 50.8 52.1 19.3 57.7 58.0 53.8 33. M.truncatula_TA25400_3880#1 46.5 43.2 35.7 37.2 41.5 41.9 43.6 45.0 45.540.4 41.5 44.1 34. P. trichocarpa_711734#1 47.0 49.6 41.0 51.0 48.1 48.748.1 48.1 17.8 51.3 53.3 48.7 35. P. trichocarpa_584053#1 51.8 56.7 46.653.7 55.4 53.4 55.7 53.1 20.2 57.0 56.0 53.4 36. M.truncatula_TA23062_3880#1 46.4 50.1 41.5 50.4 47.6 46.1 47.0 48.4 17.951.6 50.7 47.8 13 14 15 16 17 18 19 20 21 22 23 24 1. A.thaliana_AT4G14550.1#1 58.5 49.3 62.5 63.2 40.6 42.3 41.8 41.8 42.7 43.545.6 45.5 2. A. thaliana_AT3G23050.1#1 57.3 48.4 61.2 62.0 39.8 38.943.0 40.2 41.6 41.4 41.5 45.3 3. A. thaliana_AT3G23050.2#1 46.1 41.950.4 50.2 35.0 35.3 37.9 34.6 36.5 38.3 36.0 39.7 4. P.trichocarpa_566151#1 54.3 44.3 56.4 56.6 36.6 37.5 38.8 38.1 38.8 36.541.2 40.8 5. P. trichocarpa_720961#1 58.3 46.9 60.8 61.2 38.8 39.9 43.842.0 44.2 40.3 42.7 43.5 6. M. truncatula_TA20354_3880#1 61.3 50.2 64.768.0 42.2 42.8 44.5 42.7 43.5 41.8 44.6 45.8 7. S.lycopersicum_TA40922_4081#1 61.6 45.0 60.7 64.3 39.3 41.3 44.6 40.5 42.739.8 41.9 45.0 8. A. thaliana_AT1G04250.1#1 58.6 44.3 58.8 59.3 43.342.8 45.9 41.5 45.5 41.6 46.1 45.4 9. O. sativa_CB657009#1 26.9 16.424.1 24.9 20.9 22.9 21.3 20.2 20.1 19.0 20.0 21.5 10. O.sativa_TA41733_4530#1 50.0 42.0 49.8 57.0 34.9 36.6 37.9 37.5 38.9 39.942.0 43.4 11. M. truncatula_TA20951_3880#1 57.6 47.2 61.2 64.7 37.9 39.943.5 39.1 43.5 39.9 40.9 45.5 12. A. thaliana_AT3G04730.1#1 60.2 45.957.8 62.5 40.3 41.9 42.5 39.2 43.8 41.1 42.2 45.2 13. S.lycopersicum_TA48108_4081#1 45.2 58.7 60.9 43.9 46.2 47.4 44.1 47.2 43.544.9 47.2 14. M. truncatula_TA27011_3880#1 52.8 57.5 55.5 30.1 32.0 34.731.9 33.7 32.2 33.0 32.3 15. M. truncatula_TA22814_3880#1 66.9 67.6 67.739.6 43.5 43.7 42.0 42.5 40.0 41.9 41.9 16. P. trichocarpa_643213#1 70.064.5 78.0 40.1 43.5 41.8 40.4 40.6 41.3 44.4 45.6 17. A.thaliana_AT3G23030.1#1 53.4 39.5 50.6 49.4 75.0 57.5 61.7 62.4 60.7 57.560.5 18. A. thaliana_AT4G14560.1#1 56.7 41.1 50.6 52.3 85.1 60.2 60.559.7 59.8 57.2 59.0 19. A. thaliana_AT1G04240.1#1 61.5 42.8 50.6 53.268.3 69.8 62.6 65.5 59.8 57.1 58.1 20. S. lycopersicum_TA38817_4081#156.3 43.8 52.2 52.3 71.6 68.9 75.3 77.6 67.2 65.6 63.4 21. S.lycopersicum_TA43058_4081#1 60.6 43.5 51.4 52.3 68.9 67.9 75.5 84.2 66.363.3 64.9 22. P. trichocarpa_726443#1 59.1 41.5 50.6 52.3 69.8 66.7 73.480.2 77.0 83.7 68.3 23. P. trichocarpa_564913#1 60.1 41.8 51.8 56.5 65.763.8 70.0 73.9 73.4 87.0 66.2 24. P. trichocarpa_831610#1 62.0 42.8 51.857.4 69.2 68.2 73.3 74.4 76.5 79.5 74.4 25. P. trichocarpa_798526#1 61.143.8 51.0 57.0 67.3 67.3 70.4 73.9 76.4 77.4 73.9 95.0 26. M.truncatula_TA20557_3880#1 57.2 42.1 50.6 54.9 75.8 74.7 75.1 75.3 74.580.7 73.4 77.4 27. M. truncatula_TA20558_3880#1 60.1 42.1 50.2 54.0 67.268.8 74.1 77.9 75.0 78.6 74.4 80.0 28. P. trichocarpa_823671#1 62.0 44.852.2 56.5 63.5 63.1 71.9 75.4 74.9 75.9 73.9 80.3 29. P.trichocarpa_595419#1 63.0 45.2 53.9 53.2 67.7 64.2 73.1 77.6 74.6 76.672.0 81.1 30. M. truncatula_TA31746_3880#1 61.1 42.1 52.7 56.1 63.2 65.771.1 70.6 72.1 72.5 71.5 82.8 31. S. lycopersicum_TA42190_4081#1 58.744.1 51.4 55.3 68.6 71.4 75.7 72.6 74.0 75.0 67.6 76.4 32. A.thaliana_AT4G29080.1#1 49.8 51.1 54.4 55.1 42.0 41.3 47.9 44.3 44.9 45.646.9 48.5 33. M. truncatula_TA25400_3880#1 49.5 33.4 42.4 45.1 44.8 50.042.3 41.6 39.8 39.6 40.6 41.5 34. P. trichocarpa_711734#1 45.6 49.9 48.749.3 35.5 37.5 38.7 37.2 38.7 39.0 40.4 40.4 35. P. trichocarpa_584053#150.2 51.8 53.7 53.7 39.4 41.0 42.7 42.0 43.3 44.0 44.6 44.0 36. M.truncatula_TA23062_3880#1 43.8 47.0 47.3 48.7 35.7 36.3 39.8 38.9 37.839.8 38.9 41.5 25 26 27 28 29 30 31 32 33 34 35 36 1. A.thaliana_AT4G14550.1#1 43.1 42.9 44.0 43.2 41.7 44.4 42.4 43.0 36.1 38.443.3 36.5 2. A. thaliana_AT3G23050.1#1 41.8 41.9 40.7 42.6 42.4 43.842.3 43.1 33.9 39.1 43.2 37.3 3. A. thaliana_AT3G23050.2#1 36.6 37.435.0 37.4 37.3 38.2 36.6 35.7 25.0 31.7 35.1 31.5 4. P.trichocarpa_566151#1 38.6 37.9 36.5 39.7 37.9 40.4 38.3 41.8 31.5 40.041.7 37.0 5. P. trichocarpa_720961#1 43.5 42.3 41.1 44.8 42.7 43.5 42.343.3 34.8 39.5 43.6 36.6 6. M. truncatula_TA20354_3880#1 43.9 42.3 40.844.6 42.6 43.3 43.9 43.6 35.3 39.3 41.9 36.7 7. S.lycopersicum_TA40922_4081#1 42.3 39.0 41.9 42.1 42.6 44.1 41.8 43.6 37.441.5 45.9 37.6 8. A. thaliana_AT1G04250.1#1 44.6 40.8 43.2 45.9 42.444.0 42.4 42.6 35.9 37.4 41.2 36.4 9. O. sativa_CB657009#1 19.6 22.622.2 19.4 20.6 22.1 23.2 17.0 37.9 15.5 17.3 14.7 10. O.sativa_TA41733_4530#1 40.3 38.2 39.0 42.3 40.1 40.1 39.5 44.6 33.2 41.342.2 40.5 11. M. truncatula_TA20951_3880#1 43.9 40.7 39.5 44.1 43.3 42.945.5 48.0 34.9 42.4 45.0 40.9 12. A. thaliana_AT3G04730.1#1 42.9 42.442.8 41.9 43.0 41.2 44.9 42.0 35.7 37.5 41.4 36.7 13. S.lycopersicum_TA48108_4081#1 46.8 44.1 46.7 46.6 46.1 45.3 48.1 39.9 41.138.3 41.7 35.1 14. M. truncatula_TA27011_3880#1 33.0 33.7 33.3 33.9 33.835.3 35.0 33.7 26.6 31.1 33.1 30.4 15. M. truncatula_TA22814_3880#1 42.141.5 40.0 42.4 41.5 41.5 42.4 44.1 36.4 39.1 43.1 37.5 16. P.trichocarpa_643213#1 44.4 44.0 45.0 43.0 42.0 44.3 43.0 43.6 38.1 40.743.7 38.0 17. A. thaliana_AT3G23030.1#1 57.4 58.0 56.9 54.6 54.2 54.655.6 34.1 36.0 28.9 30.9 27.3 18. A. thaliana_AT4G14560.1#1 56.8 58.158.3 57.6 57.2 55.9 58.1 33.4 36.6 30.9 33.6 29.1 19. A.thaliana_AT1G04240.1#1 58.4 59.0 59.7 60.5 60.6 56.5 58.5 37.7 31.6 29.833.9 30.3 20. S. lycopersicum_TA38817_4081#1 61.9 62.6 64.2 61.8 62.461.2 59.3 35.4 31.9 29.5 33.2 30.3 21. S. lycopersicum_TA43058_4081#162.4 61.7 61.8 60.9 59.5 62.6 61.0 37.0 32.4 30.7 34.7 30.3 22. P.trichocarpa_726443#1 66.3 69.4 64.9 65.4 62.6 61.8 60.1 38.4 30.5 32.336.2 30.3 23. P. trichocarpa_564913#1 63.5 62.3 63.0 62.9 59.7 61.0 55.839.0 32.2 33.7 37.5 30.8 24. P. trichocarpa_831610#1 92.0 62.8 66.8 69.167.3 70.0 65.2 38.7 31.8 34.1 37.0 33.4 25. P. trichocarpa_798526#1 62.364.5 66.8 65.0 69.1 62.2 37.4 31.7 33.2 36.2 33.1 26. M.truncatula_TA20557_3880#1 74.9 69.4 60.9 61.0 58.8 59.0 36.7 33.2 27.832.9 29.7 27. M. truncatula_TA20558_3880#1 77.4 81.7 65.0 63.7 61.5 56.333.8 33.5 30.1 35.5 31.7 28. P. trichocarpa_823671#1 80.8 72.9 75.4 89.263.8 57.8 38.0 31.9 33.2 36.2 32.6 29. P. trichocarpa_595419#1 82.1 74.175.6 94.6 62.7 57.8 39.7 31.7 31.5 35.5 32.3 30. M.truncatula_TA31746_3880#1 82.8 73.0 73.5 76.5 77.5 60.1 38.8 31.6 33.538.4 34.8 31. S. lycopersicum_TA42190_4081#1 73.9 76.2 75.3 73.9 73.671.6 37.7 32.1 30.9 38.1 29.1 32. A. thaliana_AT4G29080.1#1 48.2 43.344.9 47.2 47.9 46.9 45.9 32.5 54.6 57.7 45.3 33. M.truncatula_TA25400_3880#1 42.2 44.9 44.1 41.9 40.8 40.2 44.3 36.7 30.736.2 28.0 34. P. trichocarpa_711734#1 39.8 36.4 36.4 39.5 39.3 40.1 39.366.8 35.2 61.4 49.1 35. P. trichocarpa_584053#1 44.6 43.0 42.0 46.3 45.046.9 46.3 69.4 39.1 69.1 47.3 36. M. truncatula_TA23062_3880#1 41.8 38.939.8 40.6 40.6 43.2 38.6 58.5 32.0 65.6 59.7

Example 4 Identification of Domains Comprised in Polypeptide SequencesUseful in Performing The Methods of the Invention 4.1. AspartateAminoTransferase (ASPAT)

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, Propom and Pfam, Smart andTIGRFAMs. Pfam is a large collection of multiple sequence alignments andhidden Markov models covering many common protein domains and families.Pfam is hosted at the Sanger Institute server in the United Kingdom.Interpro is hosted at the European Bioinformatics Institute in theUnited Kingdom.

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 4, by SEQ ID NO: 2 and by SEQ ID NO: 6 arepresented in Table D1, Table D2 and Table D3, respectively.

Tables D1, D2, D3: InterPro scan results (major accession numbers) ofthe polypeptide sequence as represented by SEQ ID NO: 4, SEQ ID NO: 2and SEQ ID NO: 6 respectively.

TABLE D1 Amino Acid Coordinates in on SEQ ID NO: 2, Database Accessionnumber Accession name (Start-End) e-value InterPro IPR000796Aspartate/other aminotransferase HMMPanther PTHR11879 ASPARTATEAMINOTRANSFERASE  [1-204] 2.6e−123 InterPro IPR004839 Aminotransferase,class I and II class HMMPfam PF00155 Aminotran_1_2 [31-203] 8.3e−61InterPro IPR015421 Pyridoxal phosphate-dependent [50-203] transferase,major region, subdomain Gene3D G3DSA:3.40.640.10 no descriptiondescription 7.8e−57 InterPro IPR015424 Pyridoxal phosphate-dependentphosphate- transferase, dependent superfamily SSF53383 PLP-dependenttransferases  [2-203] 6.2e−56

TABLE D2 Amino Acid Coordinates in Aspartate/other on SEQ ID NO: 6,Database Accession number aminotransferase (Start-End) e-value InterProIPR000796 Aspartate/other aminotransferase FPrintScan PR00799TRANSAMINASE [234-253]; [265-279]; 5.9E−68 [301-321]; [401-419];427-445] HMMPanther PTHR11879 Asp_trans  [38-460] 0.0 InterPro IPR004838Aminotransferases, class-I, pyridoxal-phosphate-binding site ProfileScanPS00105 AA_TRANSFER_CLASS_1 [303-316] 8.0E−5 InterPro IPR004839Aminotransferase, class I and II HMMPfam PF00155 Aminotran_1_2  [84-452]0.0 InterPro IPR015421 Pyridoxal phosphate-dependent transferase majorregion, subdomain I Gene3D G3DSA:3.40.640.10PyrdxlP-dep_Trfase_major_sub1 [103-375] 3.8E−111 InterPro IPR015424Pyridoxal phosphate-dependent transferase major region superfamilySSF53383 PyrdxlP-dep_Trfase_major  [55-460] 6.8E−121

TABLE D3 Amino Acid Coordinates Database Accession numberAspartate/other aminotransferase [Start-End] - Evalue InterPro IPR000796Aspartate/other aminotransferase aminoransferase FPrintScan PR00799TRANSAMINASE [179-198]; [210-224]; [246-266]; [278-303]; [346-364];[372-390]; - 1.6e−70 HMMPanther PTHR11879 ASPARTATE AMINOTRANSFERASE[1-405] - 6.2e−259 InterPro IPR004838 Aminotransferases, Class Ipyridoxal- phosphate-binding site ScanRegExp PS00105 AA_TRANSFER_CLASS_1[248-261] - 0.00008 InterPro IPR004839 Aminotransferase, class I and IIHMMPfam PF00155 Aminotran_1_2 [29-397] - 1.4e−140 InterPro IPR015421Pyridoxal phosphate-dependent transferase, major region subdomain IGene3D G3DSA:3.40.640.10 no description [48-320] - 1.7e−107 InterProIPR015424 Pyridoxal phosphate transferase major region superfamilySSF53383 PLP-dependent transferase [1-405] - 1.3e−119

4.2. MYB91 Like Transcription Factor (MYB91)

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, Propom and Pfam, Smart andTIGRFAMs. Interpro is hosted at the European Bioinformatics Institute inthe United Kingdom.

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 221 are presented in Table D4.

TABLE D4 InterPro scan results of the polypeptide sequence asrepresented by SEQ ID NO: 221 InterPro accession Integrated databaseIntegrated database Integrated database number and name Name accessionnumber accession name IPR001005 SMART SM00717 SANT SANT, DNA-bindingdomain IPR009057 homeodomain-like SUPERFAMILY SSF46689 Homeodomain-likeIPR012287 Homeodomain-related GENE3D G3DSA:1.10.10.60 IPR014778 Myb,DNA-binding PFAM PF00249 Myb_DNA-binding IPR015495 Myb transcriptionfactor PANTHER PTHR10641 MYB-related No IPR unintegrated PANTHERPTHR10641:SF24 Assymetric leaves1 and Rough Sheath2 No IPR unintegratedPROFILE PS51294 HTH_MYB

4.3. Gibberellic Acid-Stimulated Arabidopsis (GASA)

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, Propom and Pfam, Smart andTIGRFAMs. Pfam is a large collection of multiple sequence alignments andhidden Markov models covering many common protein domains and families.Pfam is hosted at the Sanger Institute server in the United Kingdom.Interpro is hosted at the European Bioinformatics Institute in theUnited Kingdom.

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 2 are presented in Table D5.

TABLE D5 InterPro scan results (major accession numbers) of thepolypeptide sequence as represented by SEQ ID NO: 276. Amino acidAccession coordinates on Database number Accession name SEQ ID NO 2InterPro IPR003854 Gibberellin regulated protein HMMPfam PF02704 GASA5-114

4.4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

The presence of conserved protein domains in SEQ ID NO: 432 wasdetermined by searching the pfam database. Pfam is a large collection ofmultiple sequence alignments and hidden Markov models covering manycommon protein domains and families. Pfam is hosted at the SangerInstitute server in the United Kingdom.

The results of the search of the Pfam with the query sequence asrepresented by SEQ ID NO: 432 are presented in Table D6.

TABLE D6 Pfam search results (major accession numbers) of thepolypeptide sequence as represented by SEQ ID NO: 432. Amino acidcoordinate of domain PF02309 in Entry SEQ ID NO: 2 HMM Bits AlignmentPfam-A Description type Start End From To score E-value mode AUX_IAAAUX/IAA Family 5 171 1 269 70.3 6.9e−18 Is family PF02309

The Alignment mode use is the so called “Is”. Parameters used in themodel are given in Table D7.

TABLE D7 HMM model Is model: hmmbuild -F HMM_Is SEED hmmcalibrate --cpu1 --seed 0 HMM_Is Is Parameter Sequence Domain Gathering cut-off −83 −83Trusted cut-off −82 −82 Noise cut-off −83.5 −83.5

4.5. IAA14 Polypeptides

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, Propom and Pfam, Smart andTIGRFAMs. Pfam is a large collection of multiple sequence alignments andhidden Markov models covering many common protein domains and families.Pfam is hosted at the Sanger Institute server in the United Kingdom.Interpro is hosted at the European Bioinformatics Institute in theUnited Kingdom.

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 738 are presented in Table D8.

TABLE D8 InterPro scan results (major accession numbers) of thepolypeptide sequence as represented by SEQ ID NO: 738. Amino acidAccession Accession coordinates on Database number name SEQ ID NO 738InterPro IPR003311 AUX/IAA protein HMMPfam PF02309 AUX_IAA  1-220InterPro IPR011525 Aux/IAA-ARF- dimerisation ProfileScan PS50962 IAA_ARF111-211 InterPro NULL NULL superfamily SSF54277 CAD & PB1 domains106-209

Example 5 Topology Prediction of the Polypeptide Sequences Useful inPerforming the Methods of the Invention 5.1. Aspartate AminoTransferase(ASPAT)

TargetP 1.1 predicts the subcellular location of eukaryotic proteins.The location assignment is based on the predicted presence of any of theN-terminal pre-sequences: chloroplast transit peptide (cTP),mitochondrial targeting peptide (mTP) or secretory pathway signalpeptide (SP). Scores on which the final prediction is based are notreally probabilities, and they do not necessarily add to one. However,the location with the highest score is the most likely according toTargetP, and the relationship between the scores (the reliability class)may be an indication of how certain the prediction is. The reliabilityclass (RC) ranges from 1 to 5, where 1 indicates the strongestprediction. TargetP is maintained at the server of the TechnicalUniversity of Denmark.

For the sequences predicted to contain an N-terminal presequence apotential cleavage site can also be predicted.

A number of parameters were selected, such as organism group (non-plantor plant), cutoff sets (none, predefined set of cutoffs, oruser-specified set of cutoffs), and the calculation of prediction ofcleavage sites (yes or no).

The protein sequences representing the GRP are used to query TargetP1.1. The “plant” organism group is selected, no cutoffs defined, and thepredicted length of the transit peptide requested.

Many other algorithms can be used to perform such analyses, including:

-   -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark

5.2. Gibberellic Acid-Stimulated Arabidopsis (GASA)

TargetP 1.1 predicts the subcellular location of eukaryotic proteins.The location assignment is based on the predicted presence of any of theN-terminal pre-sequences: chloroplast transit peptide (cTP),mitochondrial targeting peptide (mTP) or secretory pathway signalpeptide (SP). Scores on which the final prediction is based are notreally probabilities, and they do not necessarily add to one. However,the location with the highest score is the most likely according toTargetP, and the relationship between the scores (the reliability class)may be an indication of how certain the prediction is. The reliabilityclass (RC) ranges from 1 to 5, where 1 indicates the strongestprediction. TargetP is maintained at the server of the TechnicalUniversity of Denmark.

For the sequences predicted to contain an N-terminal presequence apotential cleavage site can also be predicted.

A number of parameters were selected, such as organism group (non-plantor plant), cutoff sets (none, predefined set of cutoffs, oruser-specified set of cutoffs), and the calculation of prediction ofcleavage sites (yes or no).

The results of TargetP 1.1 analysis of the polypeptide sequence asrepresented by SEQ ID NO: 221 are presented Table E1. The “plant”organism group has been selected, no cutoffs defined, and the predictedlength of the transit peptide requested. The polypeptide sequence asrepresented by SEQ ID NO: 221 is predicted to be secreted, with asecretion signal sequence of 24 amino acids.

TABLE E1 TargetP 1.1 analysis of the polypeptide sequence as representedby SEQ ID NO: 221 Length (AA) 114 Chloroplastic transit peptide 0.022Mitochondrial transit peptide 0.022 Secretory pathway signal peptide0.960 Other subcellular targeting 0.023 Predicted Location S Reliabilityclass 1 Predicted transit peptide length 24

Many other algorithms can be used to perform such analyses, including:

-   -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark    -   PSORT (URL: psort.org)    -   PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

5.3. Auxin/Indoleacetic Acid Genes (AUX/IAA)

TargetP 1.1 predicts the subcellular location of eukaryotic proteins.The location assignment is based on the predicted presence of any of theN-terminal pre-sequences: chloroplast transit peptide (cTP),mitochondrial targeting peptide (mTP) or secretory pathway signalpeptide (SP). Scores on which the final prediction is based are notreally probabilities, and they do not necessarily add to one. However,the location with the highest score is the most likely according toTargetP, and the relationship between the scores (the reliability class)may be an indication of how certain the prediction is. The reliabilityclass (RC) ranges from 1 to 5, where 1 indicates the strongestprediction. TargetP is maintained at the server of the TechnicalUniversity of Denmark.

For the sequences predicted to contain an N-terminal presequence apotential cleavage site can also be predicted.

A number of parameters were selected, such as organism group (non-plantor plant), cutoff sets (none, predefined set of cutoffs, oruser-specified set of cutoffs), and the calculation of prediction ofcleavage sites (yes or no).

Many other algorithms can be used to perform such analyses, including:

-   -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark    -   PSORT (URL: psort.org)    -   PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).        5.4. IAA14 polypeptides

TargetP 1.1 predicts the subcellular location of eukaryotic proteins.The location assignment is based on the predicted presence of any of theN-terminal pre-sequences: chloroplast transit peptide (cTP),mitochondrial targeting peptide (mTP) or secretory pathway signalpeptide (SP). Scores on which the final prediction is based are notreally probabilities, and they do not necessarily add to one. However,the location with the highest score is the most likely according toTargetP, and the relationship between the scores (the reliability class)may be an indication of how certain the prediction is. The reliabilityclass (RC) ranges from 1 to 5, where 1 indicates the strongestprediction. TargetP is maintained at the server of the TechnicalUniversity of Denmark.

For the sequences predicted to contain an N-terminal presequence apotential cleavage site can also be predicted.

A number of parameters were selected, such as organism group (non-plantor plant), cutoff sets (none, predefined set of cutoffs, oruser-specified set of cutoffs), and the calculation of prediction ofcleavage sites (yes or no).

The results of TargetP 1.1 analysis of the polypeptide sequence asrepresented by SEQ ID NO: 738 are presented Table E2. The “plant”organism group has been selected, no cutoffs defined, and the predictedlength of the transit peptide requested. The subcellular localization ofthe polypeptide sequence as represented by SEQ ID NO: 738 may be thecytoplasm or nucleus, no transit peptide is predicted.

TABLE E2 TargetP 1.1 analysis of the polypeptide sequence as representedby SEQ ID NO: 738. Name Len cTP mTP SP other Loc RC TPlen AtIAA14 2280.116 0.087 0.047 0.879 — 2 — cutoff 0.000 0.000 0.000 0.000Abbreviations: Len, Length; cTP, Chloroplastic transit peptide; mTP,Mitochondrial transit peptide, SP, Secretory pathway signal peptide,other, Other subcellular targeting, Loc, Predicted Location; RC,Reliability class; TPlen, Predicted transit peptide length.

Many other algorithms can be used to perform such analyses, including:

-   -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark    -   PSORT (URL: psort.org)    -   PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

PSORT analysis predicts a nuclear localisation, which is in agreementwith the data from the literature (Fukaki et al., 2002).

Example 6 Subcellular Localisation Prediction of the PolypeptideSequences Useful in Performing the Methods of the Invention 6.1. MYB91Like Transcription Factor (MYB91)

Experimental methods for protein localization range fromimmunolocalization to tagging of proteins using green fluorescentprotein (GFP) or beta-glucuronidase (GUS). Such methods to identifysubcellular compartmentalisation of GRF polypeptides are well known inthe art.

A predicted nuclear localisation signal (NLS) can be found by multiplesequence alignment, followed by eye inspection, in the polypeptidesequences of Table A2. An NLS is one or more short sequences ofpositively charged lysines or arginines.

Computational prediction of protein localisation from sequence data wasperformed. Among algorithms well known to a person skilled in the artare available at the ExPASy Proteomics tools hosted by the SwissInstitute for Bioinformatics, for example, PSort, TargetP, ChloroP,LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM, TMpred,and others.

The PSort algorithm predicts a nuclear subcellular localization for aMYB91 polypeptide as represented by SEQ ID NO: 221, as highestprobability (0.088). In addition, two putative NLS are predicted:

Found: pos:  81 (3) KK IAAEVPGRTA KRLGK Found: pos: 273 (3)RR VELQLESERS CRRRE

Example 7 Assay Related to the Polypeptide Sequences Useful inPerforming the Methods of the Invention 7.1. MYB91 Like TranscriptionFactor (MYB91)

MYB91 polypeptides useful in the methods of the present invention (atleast in their native form) typically, but not necessarily, havetranscriptional regulatory activity and capacity to interact with otherproteins. DNA-binding activity and protein-protein interactions mayreadily be determined in vitro or in vivo using techniques well known inthe art (for example in Current Protocols in Molecular Biology, Volumes1 and 2, Ausubel et al. (1994), Current Protocols). MYB91 polypeptidescontain two Myb DNA-binding domain (InterPro accession IPR014778).

7.2. Gibberellic Acid-Stimulated Arabidopsis (GASA)

Transgenic plants expressing GASA polypeptides (at least in their nativeform) may have enhanced tolerance to heat stress. A thermotoleranceassay is described by Ko et al. (2007): to examine the heat stress testresponse in seed germination, seeds are sown on water-saturated filterpaper. They are left to imbibe at room temperature for 18 h, transferredto 50° C., and subjected to 3 h of heat treatment. Thereafter they aretransferred to 22° C. Cotyledon emergence is determined after 5 days.Experiments are done in triplicate for each line (30 seeds each). Toassess heat tolerance assay, seeds are germinated on normal MS(Murashige & Skoog salt mixture) medium. Seven-day-old seedlings areexposed to 50° C. for 2.5 h, and the surviving plants are scored 10 daysafter returning to normal growth conditions. Experiments were done intriplicate for each line (40 seeds each). Wild type plants are used ascontrols.

7.3. IAA14 Polypeptides

IAA14 is reported to interact with ARF7 and ARF19 in a yeast two-hybridsystem (Fukaki et al., 2005): The cDNA fragments encoding the C-terminusof Arabidopsis ARF5 (amino acids 778-902), ARF7 (amino acids 1031-1164)and ARF19 (amino acids 952-1086) are amplified from a flower cDNAlibrary using the following primer sets: 5′-agaattcAATAGTAAAGGCTCATCATGGCAG-3′ and 5′-agtcgacGTTACATTTATGAAACAGAAGTCTTAAGATCG-3′ for ARF5,5′-agtcgacaAGCTCAGACTCAGCGAATGCG-3′ and 5′-cagtcgacTCACCGGTTAAACGAAGTGGC-3′ for ARF7, and 5′-gagaattcAATCAGACTCAACGAATGCG-3′ and 5′-agtcgacCTATCTGTTGAAAGAAGCTGCAGC-3′ for ARF19.

The full-length IAA14 open reading frame is amplified using two primers,5′-cgaattcAT GAACCTTAAGGAGACGGAGC-3′ and5′-tgtcgacTCATGATCTGTTCTTGAACTTCTCC-3′. PCR products are subcloned intopCR-Blunt II TOPO (Invitrogen, Carlsbad, Calif., USA) and are sequencedbefore in-frame insertion into pAD-GAL4-2.1 or pBD-GAL4 Cam (Stratagene,Calif., USA) via EcoRI/SalI (IAA14, ARF5 and ARF19) or SalI (ARF7)sites. Constructs are next introduced into Saccharomyces cerevisiae Y190cells, and transformants are subjected to assays for beta-galactosidaseactivity as previously described (Kaiser et al., Methods in YeastGenetics: A Cold Spring Harbor Laboratory Course Manual. Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, 1994).

Example 8 Cloning of the Nucleic Acid Sequence Used in the Methods ofthe Invention 8.1. Aspartate AminoTransferase (ASPAT)

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made cDNA library fromeither Arabidopsis thaliana seedlings or from Oryza sativa (in pCMVSport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi TaqDNA polymerase in standard conditions, using 200 ng of template in a 50μl PCR mix. The cDNA library and primers used are given in Table F1.

TABLE F1 Primer Primer reverse ORF in   forward (comple- SEQ ID NO:cDNA library (sense) mentary) SEQ ID NO: 3  Oryza sativa GgggacaagtttGgggaccactt gtacaaaaaagc tgtacaagaaa aggcttaaacaa gctgggtatgctggcgtcgtcgt taccatcattc cc acttca SEQ ID NO: 5 Arabidopsis GgggacaagtttGgggaccactt thaliana gtacaaaaaagc tgtacaagaaa aggcttaaacaa gctgggtaaaatggattccgtct atgtatggtcg tctctaac ctagtt SEQ ID NO: 7 ArabidopsisGgggacaagttt Ggggaccactt thaliana gtacaaaaaagc tgtacaagaaa aggcttaaacaagctgggttggt tgaaaactactc gttcagtttct atttctcttc cagac SEQ ID NO: 9Arabidopsis Ggggacaagttt Ggggaccactt thaliana gtacaaaaaagc tgtacaagaaaaggcttaaacaa gctgggttgtc tggcttctttaa atctactgaga tgttatct tggaag

Primers include the AttB sites for Gateway recombination. The amplifiedPCR fragment was purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombined in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”, pASPAT.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 1 was then used in an LR reactionwith a destination vector used for Oryza sativa transformation. Thisvector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 218) for constitutive specific expression waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::ASPAT (FIG. 3) was transformed into Agrobacterium strain LBA4044according to methods well known in the art.

Similarly, expression vectors were generated comprising the followingfeatures (Table F2):

TABLE F2 ASPT nucleic acid (Longest Vector Promoter ORF in SEQ ID NO:)ExprVect1 pPR (SEQ ID NO: 219) SEQ ID NO: 3 ExprVect2 pGOS2 (SEQ ID NO:218) SEQ ID NO: 5 ExprVect3 pPR (SEQ ID NO: 219) SEQ ID NO: 5 ExprVect4pGOS2 (SEQ ID NO: 218) SEQ ID NO: 7 ExprVect5 pGOS2 (SEQ ID NO: 218) SEQID NO: 9

8.2. MYB91 Like Transcription Factor (MYB91)

Unless otherwise stated, recombinant DNA techniques are performedaccording to standard protocols described in (Sambrook (2001) MolecularCloning: a laboratory manual, 3rd Edition Cold Spring Harbor LaboratoryPress, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994),Current Protocols in Molecular Biology, Current Protocols. Standardmaterials and methods for plant molecular work are described in PlantMolecular Biology Labfax (1993) by R. D. D. Croy, published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications(UK).

The Populus trichocarpa nucleic acid sequence encoding a MYB91polypeptide sequence as represented by SEQ ID NO: 221 was amplified byPCR using as template a cDNA bank constructed using RNA from tomatoplants at different developmental stages. The following primers, whichinclude the AttB sites for Gateway recombination, were used for PCRamplification:

1) prm11884 (SEQ ID NO: 271, sense):5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGAAGGAGA GGCAGCGT-3′2) prm11885 (SEQ ID NO: 272, reverse, complementary):5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGACCTGATACAGCTGG ACGTA-3′

PCR was performed using Hifi Taq DNA polymerase in standard conditions.A PCR fragment of the expected length (including attB sites) wasamplified and purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombined in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”. PlasmidpDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 220 was subsequently used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 53) for constitutive expression was locatedupstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::MYB91 (FIG. 6) for constitutive expression, was transformed intoAgrobacterium strain LBA4044 according to methods well known in the art.

8.3. Gibberellic Acid-Stimulated Arabidopsis (GASA)

a) Cloning of Tomato GASA:

The tomato nucleic acid sequence used in the methods of the inventionwas amplified by PCR using as template a custom-made Solanumlycopersicum seedlings cDNA library (in pCMV Sport 6.0; Invitrogen,Paisley, UK). PCR was performed using Hifi Taq DNA polymerase instandard conditions, using 200 ng of template in a 50 μl PCR mix. Theprimers used were prm10623 (SEQ ID NO: 286; sense, start codon in bold):5′-ggggacaagtttgtacaaaaaagc aggcttaaacaatggagaagacacttagctta-3′ andprm10624 (SEQ ID NO: 287; reverse, complementary):5′-ggggaccactttgtacaagaaagctgggtatatatgattaagggcatttt-3′, which includethe AttB sites for Gateway recombination. The amplified PCR fragment waspurified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombined in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”, pGASA. PlasmidpDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 275 was then used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 290) for constitutive specific expression waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::GASA (FIG. 3) was transformed into Agrobacterium strain LBA4044according to methods well known in the art.

b) Cloning of Poplar GASA

The poplar nucleic acid sequence used in the methods of the inventionwas amplified by PCR using as template a custom-made poplar seedlingscDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR wasperformed using Hifi Taq DNA polymerase in standard conditions, using200 ng of template in a 50 μl PCR mix. The primers used were prm10625(SEQ ID NO: 288; sense, start codon in bold):5′-gggacaagtttgtacaaaaaagcaggctt aacaatgaagaagctcttctttgct-3′ andprm10626 (SEQ ID NO: 289; reverse, complementary):5′-ggggaccactttgtacaagaaagctgggtacatgcacatcttgactgtct-3′, which includethe AttB sites for Gateway recombination. The amplified PCR fragment waspurified also using standard methods, and the further cloning procedurewas as described above, including use of the rice GOS2 promoter.

8.4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Oryza sativa seedlingscDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR wasperformed using Hifi Taq DNA polymerase in standard conditions, using200 ng of template in a 50 μl PCR mix with a set of primer complementaryto the first and last 20 nucleotides of SEQ ID NO: 431. The sequence ofthe forward primer used in the PCR can be represented by SEQ ID NO: 667and the reverse primer by SEQ ID NO: 668. The amplified PCR fragment waspurified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombined in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”, p AUX/IAA.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 431 was then used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 669) for constitutive specific expression waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vector pGOS2::AUX/IAA (FIG. 12) was transformed into Agrobacterium strain LBA4044according to methods well known in the art.

8.5. IAA14 Polypeptides

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Arabidopsis thalianaseedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCRwas performed using Hifi Taq DNA polymerase in standard conditions,using 200 ng of template in a 50 μl PCR mix. The primers used wereprm07273 (SEQ ID NO: 745; sense, start codon in bold):5′-ggggacaagtttgtacaaaaaagcagg cttaaacaatgaaccttaaggagacggag-3′ andprm07274 (SEQ ID NO: 746; reverse, complementary):5′-ggggaccactttgtacaagaaagctgggttcaatgcatattgtcctctttt-3′, which includethe AttB sites for Gateway recombination. The amplified PCR fragment waspurified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombined in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”, pIAA14-like.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 737 was then used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice HMGPpromoter (SEQ ID NO: 747) for weak constitutive expression was locatedupstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpHMGP::IAA14-like (FIG. 16) was transformed into Agrobacterium strainLBA4044 according to methods well known in the art.

Example 9 Plant Transformation Rice Transformation

The Agrobacterium containing the expression vector was used to transformOryza sativa plants. Mature dry seeds of the rice japonica cultivarNipponbare were dehusked. Sterilization was carried out by incubatingfor one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂,followed by a 6 times 15 minutes wash with sterile distilled water. Thesterile seeds were then germinated on a medium containing 2,4-D (callusinduction medium). After incubation in the dark for four weeks,embryogenic, scutellum-derived calli were excised and propagated on thesame medium. After two weeks, the calli were multiplied or propagated bysubculture on the same medium for another 2 weeks. Embryogenic calluspieces were sub-cultured on fresh medium 3 days before co-cultivation(to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

Approximately 35 independent T0 rice transformants were generated forone construct. The primary transformants were transferred from a tissueculture chamber to a greenhouse. After a quantitative PCR analysis toverify copy number of the T-DNA insert, only single copy transgenicplants that exhibit tolerance to the selection agent were kept forharvest of T1 seed. Seeds were then harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al.1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M patent U.S. Pat. No. 5,164,310. Severalcommercial soybean varieties are amenable to transformation by thismethod. The cultivar Jack (available from the Illinois Seed foundation)is commonly used for transformation. Soybean seeds are sterilised for invitro sowing. The hypocotyl, the radicle and one cotyledon are excisedfrom seven-day old young seedlings. The epicotyl and the remainingcotyledon are further grown to develop axillary nodes. These axillarynodes are excised and incubated with Agrobacterium tumefacienscontaining the expression vector. After the cocultivation treatment, theexplants are washed and transferred to selection media. Regeneratedshoots are excised and placed on a shoot elongation medium. Shoots nolonger than 1 cm are placed on rooting medium until roots develop. Therooted shoots are transplanted to soil in the greenhouse. T1 seeds areproduced from plants that exhibit tolerance to the selection agent andthat contain a single copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown DCW and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Cotton Transformation

Cotton is transformed using Agrobacterium tumefaciens according to themethod described in U.S. Pat. No. 5,159,135. Cotton seeds are surfacesterilised in 3% sodium hypochlorite solution during 20 minutes andwashed in distilled water with 500 μg/ml cefotaxime. The seeds are thentransferred to SH-medium with 50 μg/ml benomyl for germination.Hypocotyls of 4 to 6 days old seedlings are removed, cut into 0.5 cmpieces and are placed on 0.8% agar. An Agrobacterium suspension (approx.108 cells per ml, diluted from an overnight culture transformed with thegene of interest and suitable selection markers) is used for inoculationof the hypocotyl explants. After 3 days at room temperature andlighting, the tissues are transferred to a solid medium (1.6 g/lGelrite) with Murashige and Skoog salts with B5 vitamins (Gamborg etal., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/l 2,4-D, 0.1 mg/l6-furfurylaminopurine and 750 μg/ml MgCL2, and with 50 to 100 μg/mlcefotaxime and 400-500 μg/ml carbenicillin to kill residual bacteria.Individual cell lines are isolated after two to three months (withsubcultures every four to six weeks) and are further cultivated onselective medium for tissue amplification (30° C., 16 hr photoperiod).Transformed tissues are subsequently further cultivated on non-selectivemedium during 2 to 3 months to give rise to somatic embryos. Healthylooking embryos of at least 4 mm length are transferred to tubes with SHmedium in fine vermiculite, supplemented with 0.1 mg/l indole aceticacid, 6 furfurylaminopurine and gibberellic acid. The embryos arecultivated at 30° C. with a photoperiod of 16 hrs, and plantlets at the2 to 3 leaf stage are transferred to pots with vermiculite andnutrients. The plants are hardened and subsequently moved to thegreenhouse for further cultivation.

Example 10 Phenotypic Evaluation Procedure 10.1 Evaluation Setup

Approximately 35 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Events, of which the T1progeny segregated 3:1 for presence/absence of the transgene, wereretained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%. Plantsgrown under non-stress conditions were watered at regular intervals toensure that water and nutrients were not limiting and to satisfy plantneeds to complete growth and development.

T1 events were further evaluated in the T2 generation following the sameevaluation procedure as for the T1 generation but with more individualsper event. From the stage of sowing until the stage of maturity theplants were passed several times through a digital imaging cabinet. Ateach time point digital images (2048×1536 pixels, 16 million colours)were taken of each plant from at least 6 different angles.

Drought Screen

Plants from T2 seeds are grown in potting soil under normal conditionsuntil they approached the heading stage. They are then transferred to a“dry” section where irrigation is withheld. Humidity probes are insertedin randomly chosen pots to monitor the soil water content (SWC). WhenSWC goes below certain thresholds, the plants are automaticallyre-watered continuously until a normal level is reached again. Theplants are then re-transferred again to normal conditions. The rest ofthe cultivation (plant maturation, seed harvest) is the same as forplants not grown under abiotic stress conditions. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Nitrogen Use Efficiency Screen

Rice plants from T2 seeds are grown in potting soil under normalconditions except for the nutrient solution. The pots are watered fromtransplantation to maturation with a specific nutrient solutioncontaining reduced N nitrogen (N) content, usually between 7 to 8 timesless. The rest of the cultivation (plant maturation, seed harvest) isthe same as for plants not grown under abiotic stress. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Salt Stress Screen

Plants are grown on a substrate made of coco fibers and argex (3 to 1ratio). A normal nutrient solution is used during the first two weeksafter transplanting the plantlets in the greenhouse. After the first twoweeks, 25 mM of salt (NaCl) is added to the nutrient solution, until theplants are harvested. Seed-related parameters are then measured.

10.2 Statistical Analysis: F Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.

The F test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF test. A significant F test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

Because two experiments with overlapping events were carried out, acombined analysis was performed. This is useful to check consistency ofthe effects over the two experiments, and if this is the case, toaccumulate evidence from both experiments in order to increaseconfidence in the conclusion. The method used was a mixed-model approachthat takes into account the multilevel structure of the data (i.e.experiment—event—segregants). P values were obtained by comparinglikelihood ratio test to chi square distributions.

10.3 Parameters Measured Biomass-Related Parameter Measurement

From the stage of sowing until the stage of maturity the plants werepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles.

The plant aboveground area (or leafy biomass) was determined by countingthe total number of pixels on the digital images from aboveground plantparts discriminated from the background. This value was averaged for thepictures taken on the same time point from the different angles and wasconverted to a physical surface value expressed in square mm bycalibration. Experiments show that the aboveground plant area measuredthis way correlates with the biomass of plant parts above ground. Theabove ground area is the area measured at the time point at which theplant had reached its maximal leafy biomass. The early vigour is theplant (seedling) aboveground area three weeks post-germination. Increasein root biomass is expressed as an increase in total root biomass(measured as maximum biomass of roots observed during the lifespan of aplant); or as an increase in the root/shoot index (measured as the ratiobetween root mass and shoot mass in the period of active growth of rootand shoot).

Early vigour was determined by counting the total number of pixels fromaboveground plant parts discriminated from the background. This valuewas averaged for the pictures taken on the same time point fromdifferent angles and was converted to a physical surface value expressedin square mm by calibration. The results described below are for plantsthree weeks post-germination.

Seed-Related Parameter Measurements

The mature primary panicles were harvested, counted, bagged,barcode-labelled and then dried for three days in an oven at 37° C. Thepanicles were then threshed and all the seeds were collected andcounted. The filled husks were separated from the empty ones using anair-blowing device. The empty husks were discarded and the remainingfraction was counted again. The filled husks were weighed on ananalytical balance. The number of filled seeds was determined bycounting the number of filled husks that remained after the separationstep. The total seed yield was measured by weighing all filled husksharvested from a plant. Total seed number per plant was measured bycounting the number of husks harvested from a plant. Thousand KernelWeight (TKW) is extrapolated from the number of filled seeds counted andtheir total weight. The Harvest Index (HI) in the present invention isdefined as the ratio between the total seed yield and the above groundarea (mm²), multiplied by a factor 10⁶. The total number of flowers perpanicle as defined in the present invention is the ratio between thetotal number of seeds and the number of mature primary panicles. Theseed fill rate as defined in the present invention is the proportion(expressed as a %) of the number of filled seeds over the total numberof seeds (or florets).

Examples 11 Results of the Phenotypic Evaluation of the TransgenicPlants 11.1. Aspartate AminoTransferase (ASPAT)

The results of the evaluation of transgenic rice plants in the T2generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 3 under the control of the rice GOS2promoter in non-stress conditions are presented below (Table G1). Seeprevious Examples for details on the generations of the transgenicplants. An increase of at least 5% was observed for aboveground biomass(AreaMax), emergence, seed yield (totalwgseeds), number of filled seeds(nrfilledseed), fill rate (fillrate), and plant height (HeightMax)(Table G1).

TABLE G1 Phenotype transgenic plants transformed with pGOS2::ASAPT. %increase in transgenic plants Parameter versus control plants AreaMax7.4 totalwgseeds 11.8 nrfilledseed 9.3 fillrate 5.0 HeightMax 5.0

The results of the evaluation of transgenic rice plants in the T1generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 5 under the control of the rice GOS2promoter in non-stress conditions are presented below (Table G2). Seeprevious Examples for details on the generations of the transgenicplants. An increase of at least 5% was observed for plant height(HeightMax).

TABLE G2 Phenotype transgenic plants transformed with ExprVect2. %increase in transgenic plants Parameter versus control plants Plantheigth 5.2

The results of the evaluation of transgenic rice plants in the T1generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 5 under the control of the rice PR promoterin non-stress conditions are presented below (Table G3). See previousExamples for details on the generations of the transgenic plants. Anincrease of at least 5% was observed for aboveground biomass (AreaMax),emergence vigour (EmerVigor), seed yield (totalwgseeds), number offilled seeds (nrfilledseed), number of flowers per panicle(flowerperpan), number of first panicle (firstpan), total number ofseeds (nrtotalseed) and plant height (HeightMax).

TABLE G3 Phenotype transgenic plants transformed with the expressionvector ExprVect3. % increase in transgenic plants Parameter versuscontrol plants AreaMax 29.3 EmerVigor 49.8 totalwgseeds 31.2nrfilledseed 32.0 flowerperpan 9.5 firstpan 15.8 nrtotalseed 26.8HeightMax 11.6

The results of the evaluation of transgenic rice plants in the T2generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 5 under the control of the rice PR promoterin non-stress conditions are presented below (Table G4). See previousExamples for details on the generations of the transgenic plants. Anincrease of at least 5% was observed for aboveground biomass (AreaMax),emergence vigour (EmerVigor), total seed yield (totalwgseeds), number offilled seeds (nrfilledseed), nr of flowers per panicle (flowerperpan),number of first panicle (firstpan), total number of seeds (nrtotalseed)and plant height (HeightMax).

TABLE G4 Phenotype transgenic plants transformed with the expressionvector ExprVect3. % increase in transgenic plants Parameter versuscontrol plants AreaMax 9.7 EmerVigor 17.8 totalwgseeds 24.4 nrfilledseed23.3 fillrate 8.4 harvestindex 14.7 firstpan 10.8 nrtotalseed 14.9HeightMax 5.3

The results of the evaluation of transgenic rice plants in the T1generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 3 under the control of the rice PR promoterin non-stress conditions are presented below (Table G5). See previousExamples for details on the generations of the transgenic plants. Anincrease of at least 5% was observed for seed yield (totalwgseeds),number of filled seeds (nrfilledseed), harvest index (harvestindex), andseed filling rate (fillrate).

TABLE G5 Phenotype transgenic plants transformed with the expressionvector ExprVect1. % increase in transgenic plants Parameter versuscontrol plants totalwgseeds 23.0 nrfilledseed 20.1 fillrate 9.9harvestindex 13.8

The results of the evaluation of transgenic rice plants in the T1generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 9 under the control of the rice GOS2promoter in non-stress conditions are presented below (Table G6). Seeprevious Examples for details on the generations of the transgenicplants. An increase of at least 5% was observed for filled seeds(nrfilledseed) and harvest index (harvestindex).

TABLE G6 Phenotype transgenic plants transformed with the expressionvector ExprVect5. % increase in transgenic plants Parameter versuscontrol plants fillrate 6.6 harvestindex 6.0

The results of the evaluation of transgenic rice plants under non-stressconditions are presented below. An increase of at least 5% was observedfor fill rate and harvest index.

11.2. MYB91 Like Transcription Factor (MYB91)

The results of the evaluation of T1 generation transgenic rice plantsexpressing the nucleic acid sequence encoding a MYB91 polypeptide asrepresented by SEQ ID NO: 221, under the control of a constitutivepromoter, and grown under normal growth conditions, are presented below.

There was a significant increase in plant height, in harvest index (HI),and in Thousand Kernel Weight (TKW).

TABLE G7 Results of the evaluation of T1 generation transgenic riceplants expressing the nucleic acid sequence encoding a MYB91 polypeptideas represented by SEQ ID NO: 221, under the control of a promoter forconstitutive expression. 0verall average % increase Trait in 4 events inthe T2 generation Plant height 3% Harvest index 8% Thousand kernelweight 6%

11.3. Gibberellic Acid-Stimulated Arabidopsis (GASA)

The results of the evaluation of transgenic rice plants expressing thetomato GASA nucleic acid under control of a medium strength constitutivepromoter under non-stress conditions are presented below in Table G8.

TABLE G8 overall increase (%) for yield parameters parameter 1^(st)evaluation 2^(nd) evaluation Time to flower 2.1 3.5 Fill rate 10.4 8.3Flowers per panicle 4.8 14.7

The flowering time was reduced compared to control plants, and there wasan increase of more than 5% for fill rate and for the number of flowersper panicle.

The results of the evaluation of transgenic rice plants expressing thepoplar GASA nucleic acid under control of a medium strength constitutivepromoter under non-stress conditions are presented below in Table G9.

TABLE G9 overall increase (%) for yield parameters parameter 1^(st)evaluation 2^(nd) evaluation Total weight of seeds 13.3 13.7 Harvestindex 18.8 22.2 Thousand Kernel weight 4.2 2.9

11.4. Auxin/Indoleacetic Acid Genes (AUX/IAA)

The results of the evaluation of transgenic rice plants in the T2generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 431 under non-stress conditions arepresented below. See previous Examples for details on the generations ofthe transgenic plants.

The results of the evaluation of transgenic rice plants under non-stressconditions are presented below (Table G10). An increase of at least 5%was observed for the number of filled seed per plant (nrfilledseed),harvest index (harvestindex) and seed yield (totalwgseeds.

TABLE G10 Percentage increase in transgenic Yield-related trait plantscompared to control plants totalwgseeds 12.0 harvestindex 8.3nrfilledseed 11.2

11.5. IAA14 Polypeptides

The results of the evaluation of T2 transgenic rice plants expressingthe IAA14-like nucleic acid of SEQ ID NO: 738 under non-stressconditions are presented below (Table G11).

TABLE G11 Overall yield increase (in %) of transgenic plants expressingSEQ ID NO: 738 Parameter Overall increase totalwgseeds 19.2 nrfilledseed18.6 fillrate 18.8 harvestindex 21.1 HeightMax 5.5 GravityYMax 6.6

An increase was found for total weight of seeds, the number of filledseeds, for the fill rate (number of filled seeds divided by the totalnumber of seeds and multiplied by 100), harvest index, height of theplant and the gravity center (indication of branching of plants). Foreach of the parameters listed in Table G11, the p-value was p<0.05.

1. A method for enhancing yield-related traits in plants relative tocontrol plants, comprising modulating expression in a plant of a nucleicacid encoding an ASPAT (Aspartate Aminotransferase) polypeptidecomprising an Aminotransferase class I and II (Aminotran_(—)1_(—)2)domain (Interpro accession number: IPR004839; pfam accession number:PF00155), and optionally selecting plants having enhanced yield-relatedtraits.
 2. The method of claim 1, wherein said ASPAT polypeptidecomprises one or more motifs having at least 50% identity to any one ormore of the following motifs: (i) Motif 1: NPTG, (SEQ ID NO: 207) (ii)Motif 2: IVLLHACAHNPTGVDPT, (SEQ ID NO: 208) and (iii)Motif 3: SRLLILCSPSNPTGSVY. (SEQ ID NO: 209)

wherein any amino acid residue maybe substituted by a conserved aminoacid.
 3. The method of claim 1, wherein said modulated expression iseffected by introducing and expressing in a plant the nucleic acidencoding an ASPAT polypeptide.
 4. The method of claim 1, wherein saidnucleic acid encoding an ASPAT polypeptide encodes any one of theproteins listed in Table A or is a portion of such a nucleic acid, or anucleic acid capable of hybridising with such a nucleic acid.
 5. Themethod of claim 1, wherein said nucleic acid encoding an ASPATpolypeptide encodes an orthologue or paralogue of any of the proteinsgiven in Table A.
 6. The method of claim 1, wherein said enhancedyield-related traits comprise increased yield, increased biomass, and/orincreased seed yield relative to control plants.
 7. The method of claim1, wherein said enhanced yield-related traits are obtained undernon-stress conditions.
 8. The method of claim 1, wherein said enhancedyield-related traits are obtained under conditions of drought stress,salt stress or nitrogen deficiency.
 9. The method of claim 3, whereinsaid nucleic acid encoding an ASPAT polypeptide is operably linked to aconstitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice.10. The method of claim 3, wherein said nucleic acid encoding an ASPATpolypeptide is operably linked to a green tissue-specific promoter, a PRpromoter, or a PR promoter from rice.
 11. The method of claim 1, whereinsaid nucleic acid encoding an ASPAT polypeptide is of plant origin, froma dicotyledonous plant, from the family poaceae, from the genus Oryza,or from Oryza sativa.
 12. A plant or part thereof, including seeds,obtained by the method of claim 1, wherein said plant or part thereofcomprises a recombinant nucleic acid encoding an ASPAT polypeptide. 13.A construct comprising: (i) the nucleic acid encoding an ASPATpolypeptide as defined in claim 1; (ii) one or more control sequencescapable of driving expression of the nucleic acid of (a); and optionally(iii) a transcription termination sequence.
 14. The construct of claim13, wherein one of said control sequences is a constitutive promoter, aGOS2 promoter, or a GOS2 promoter from rice.
 15. The construct of claim13, wherein one of said control sequences is a green tissue-specificpromoter, a PR promoter, or a PR promoter from rice.
 16. A method formaking plants having increased yield, increased biomass, and/orincreased seed yield relative to control plants, comprising transformingthe construct of claim 13 into a plant.
 17. A plant, plant part or plantcell transformed with the construct of claim
 13. 18. A method for theproduction of a transgenic plant having increased yield, increasedbiomass, and/or increased seed yield relative to control plants,comprising: (i) introducing and expressing in a plant the nucleic acidencoding an ASPAT polypeptide as defined in claim 1; and (ii)cultivating the plant cell under conditions promoting plant growth anddevelopment.
 19. A transgenic plant having increased yield, increasedbiomass, and/or increased seed yield, relative to control plants,resulting from modulated expression of the nucleic acid encoding anASPAT polypeptide as defined in claim 1, or a transgenic plant cellderived from said transgenic plant.
 20. The transgenic plant of claim17, or a transgenic plant cell derived thereof, wherein said plant is acrop plant or a monocot or a cereal, such as rice, maize, wheat, barley,millet, rye, triticale, sorghum emmer, spelt, secale, einkom, teff,milo, and oats.
 21. Harvestable parts of the transgenic plant of claim20, wherein said harvestable parts are shoot biomass and/or seeds. 22.Products derived from the transgenic plant of claim 20 and/or fromharvestable parts of of said transgenic plant.
 23. (canceled)
 24. Anisolated nucleic acid molecule selected from: (i) a nucleic acidrepresented by any one of SEQ ID NO: 81, 147, 153, 183 and 185; (ii) thecomplement of a nucleic acid represented by any one of SEQ ID 81, 147,153, 183 and 185; (iii) a nucleic acid encoding the polypeptide asrepresented by any one of SEQ ID NO: 82, 148, 154, 184 and 186 as aresult of the degeneracy of the genetic code, said nucleic acid can bederived from a polypeptide sequence as represented by any one of SEQ IDNO: 82, 148, 154, 184 and 186 and confers enhanced yield-related traitsrelative to control plants; (iv) a nucleic acid having at least 30%sequence identity with any of the nucleic acid sequences of Table A andconferring enhanced yield-related traits relative to control plants; (v)a nucleic acid which hybridizes with the nucleic acid of (i) to (iv)under stringent hybridization conditions and confers enhancedyield-related traits relative to control plants; (vi) a nucleic acidencoding an ASPAT polypeptide having at least 50% sequence identity tothe amino acid sequence represented by any one of SEQ ID NO: 82, 148,154, 184 and 186 and any of the other amino acid sequences in Table Aand conferring enhanced yield-related traits relative to control plants.25. An isolated polypeptide selected from: (i) an amino acid sequencerepresented by any one of SEQ ID NO: 82, 148, 154, 1184 and 186; (ii) anamino acid sequence having at least 50% sequence identity to the aminoacid sequence represented by any one of SEQ ID NO: 82, 148, 154, 184 and186, and any of the other amino acid sequences in Table A and conferringenhanced yield-related traits relative to control plants. (iii)derivatives of any of the amino acid sequences given in (i) or (ii)above.